WO2026008664A1 - Allosteric modulators of inhibitory immune receptor complexes - Google Patents

Abstract

This disclosure relates to an immunoglobulin single variable domain (ISVD)-containing modulator that allosterically binds to a three-dimensional (3D) epitope of a protein complex comprising at least one inhibitory immune receptor and at least one corresponding ligand. The modulator regulates cooperativity within such complexes, thereby affecting the binding affinity and downstream signaling pathways. Specifically, the allosteric modulator of this invention induces positive cooperativity in immunoinhibitory receptor-ligand complexes, thus suppressing immune responses in a spatiotemporally restricted manner. Accordingly, these allosteric modulators are useful as therapeutic agents for inflammatory diseases, such as autoimmune diseases, allergic diseases, or graft-versus-host disease (GVHD). Moreover, this disclosure pertains to methods for identifying, selecting, and producing said allosteric modulators.

Description

ALLOSTERIC MODULATORS OF INHIBITORY IMMUNE RECEPTOR

COMPLEXES

FIELD OF THE INVENTION

This disclosure relates to an immunoglobulin single variable domain (ISVD)-containing modulator that allosterically binds to a three-dimensional (3D) epitope of a protein complex comprising at least one inhibitory immune receptor and at least one corresponding ligand. The modulator regulates cooperativity within such complexes, thereby affecting the binding affinity and downstream signaling pathways. Specifically, the allosteric modulator of this invention induces positive cooperativity in immunoinhibitory receptor-ligand complexes, thus suppressing immune responses in a spatiotemporally restricted manner. Accordingly, these allosteric modulators are useful as therapeutic agents for inflammatory diseases, such as autoimmune diseases, allergic diseases, or graft-versus-host disease (GVHD). Moreover, this disclosure pertains to methods for identifying, selecting, and producing said allosteric modulators.

BACKGROUND

A 2023 population-based study on 22 million individuals demonstrated that approximately one in ten people are afflicted with at least one of the 19 most prevalent autoimmune diseases, examples of which include rheumatoid arthritis, psoriasis, Hashimoto's thyroiditis, coeliac disease, type 1 diabetes and systemic lupus erythematosus¹. Autoimmune disorders represent a heterogeneous group of conditions characterized by abnormal T cell and B cell responses to the body's own components, i.e., autoantigens. Such responses, termed “autoreactive”, stem from both central and peripheral defects in tolerance checkpoints and are triggered by environmental factors (such as infectious agents), molecular mimicry and/or genetic predispositions^2-5.

Current therapeutic approaches for autoimmune diseases rely on immunosuppressive agents which broadly dampen immune responses. The main categories of such drugs include inhibitors of TNF and JAK, antagonists of IL-6R, IL-12, IL-17A and IL-23, B cell-depleting agents, integrin blockers and inhibitors of co-stimulatory molecules⁶. While these therapies are considered the “gold standard” of care, they usually necessitate long-term usage of high drug doses to sustain disease control. Extended exposure to such medications can render patients susceptible to life-threatening opportunistic infections and elevate the risk of developing cancer. Moreover, the benefits of these drugs are often offset by their toxicity and serious side effects⁷. Notably, it is evident that the majority of patients either do not respond optimally or fail to respond altogether to currently available immunosuppressants⁶. Therefore, there is a critical need for more targeted therapeutic approaches for autoimmunity, aiming to minimize the risk of systemic immune suppression and improve patient tolerability.

An inhibitory immune receptor transmits intracellular signaling upon binding with its orthosteric ligand(s), leading to the formation of an immune complex, such as PD-1 »PD-L1 , PD-1 »PD-L2 or CTLA4»B7.1 . In general, immune checkpoint complexes form in trans between receptor-expressing T cells and ligand-expressing antigen-presenting cells (immune cells or cancer cells), creating a milieu known as the “immunological synapse”. When the inhibitory or stimulatory checkpoint receptors bind with their ligands, they mediate an immunosuppressive or immunoactivating signal to T cells, respectively.

The co-inhibitory pathways of T cells are often hijacked by tumors, resulting in T cell exhaustion, attenuated effector functions and failure to control cancer progression⁸. Consequently, blockade of inhibitory immune checkpoints has been proven effective in the treatment of a variety of tumor types, including melanoma, non-small cell lung carcinoma, colorectal cancer, and hepatocellular carcinoma⁹. Interestingly, a considerable number of patients with no previous history of autoimmunity, who undergo treatment with such blockers, develop autoimmune side effects¹⁰. This observation aligns with a growing recognition of the significance of immune receptors in the pathogenesis of autoimmunity. A number of reports indicate that agonism of inhibitory immune receptors, aimed at enhancing immunosuppressive signaling, could potentially offer therapeutic benefits in managing autoimmunity^11-13. This concept is being explored by several preclinical and clinical programs.

A number of inhibitory checkpoint receptors have been studied in this context, with PD-1 emerging as the most exploited target, owing to its fundamental role in T cell exhaustion¹⁴. Current clinical PD-1 agonist programs utilize monoclonal antibody approaches aimed at directly stimulating the receptor, and include: i) Peresolimab (WO2019/168745)¹⁵, ii) Rosnilimab (WO2020/247648)¹⁶, iii) JNJ-4703 (WO2018/226580) and GS-0151 (WO2023/089377). These antibodies are at various stages of development for several autoimmune indications, such as rheumatoid arthritis (RA), colitis, atopic dermatitis and Sjogren syndrome. Besides, several other immunoinhibitory receptor agonists are being tested in preclinical models of autoreactivity. Nevertheless, broad immune suppression associated with these approaches may lead to serious adverse effects, including poor response to vaccinations, impaired viral clearance, and heightened susceptibility to cancer¹¹. Moreover, chronic exposure to such agonists may result in receptor desensitization and decreased drug responsiveness overtime¹⁷.

Notably, Wang et al. demonstrated that biorthogonal immobilization of PD-L1 (the primary orthosteric ligand of PD-1 ) on the cell surface, achieved through a two-step dual-anchor coupling strategy, resulted in prolonged presence of PD-L1 on target cells, which supported the formation of PD-1 »PD-L1 complexes¹⁸. The subsequent PD-1 receptor activation elicited therapeutic effects in murine models of autoimmune diabetes and arthritis. Along a similar conceptual framework, several preclinical studies showcased potential benefits of administering PD-L1 -overexpressing exogenous cells or extracellular vesicles derived from such cells. This approach has demonstrated efficacy in reducing the infiltration of autoreactive T cells while promoting the recruitment of immunosuppressive regulatory T cells (Tregs)^19-21. Furthermore, Sugiura et al. showed that facilitating PD-1 »PD-L1 interaction by sequestering PD-L1 from the cis-PD-L1 »CD80 duplex effectively alleviated symptoms of several autoimmune diseases in mice²². Overall, these findings support the potential of selectively tuning immunoinhibitory complexes as a viable therapeutic approach for autoreactive conditions.

Protein complexformation is essentialto numerous biological processes and is implicated in many human diseases, representing a significant target potential for modern drug design. Traditionally, strategies for modulating protein-protein interactions (PPIs) have focused on inhibitory modalities (e.g., blockers, antagonists)²³. However, recent advances in the field have underscored the promise of therapeutic PPI stabilization^24-26. Notable examples of PPI stabilizers include immunosuppressants such as cyclosporin (Sandimmun®, Novartis Pharmaceuticals), FK506 (Prograf®, Astellas Pharma), and rapamycin (Rapamune®, Pfizer). Yet, the discoveries of such stabilizers have predominantly been serendipitous²⁴²⁷. Unlike the binding pockets typically found in conventional protein targets, PPI interfaces tend to be flat. Consequently, classic medicinal chemistry methods are less effective for designing and identifying PPIs modulators²⁶. Thus, rational and systematic approaches are necessary to guide the development of new PPI stabilizers.

In this context. WQ2020/205626 describes methods for generating affinity and activity modulators of interactions between various extracellular proteins, including immune receptors and their ligands, said modulators specifically binding only one subunit (i.e., a receptor molecule or a ligand molecule) of such protein complexes. Moreover, Nanobodies™ (Nbs) that inhibit or facilitate the formation of the SOS1 »RAS complex were generated²⁸ utilizing the “Cross-linked PPIs and immunize llamas (ChILL)” and “Display and co-selection (DisCO)” methods as disclosed in WQ2016/012363. However, the clinical significance of these nanobodies has yet to be evaluated. In sum, effective management and treatment of autoimmune diseases are widely acknowledged as major unmet medical needs. Established therapeutic strategies and those under clinical development generally lack specificity, pose toxicity risks, and compromise user safety. In light of the above, there is a clear imperative to develop selective modulators of inhibitory immune receptor complexes, particularly to deliver innovative therapeutic solutions for treating autoimmune diseases. Such selective modulators could also exert therapeutic effects in the treatment of other inflammatory conditions, including graft-versus-host disease (GVHD) and allergic diseases.

SUMMARY OF THE INVENTION

The present invention addresses the aforementioned challenges by introducing novel positive allosteric modulators (PAMs) of immunoinhibitory complexes.

In one aspect, the allosteric modulators disclosed herein improve the binding affinity within immunoinhibitory receptor-ligand complexes through positive modulation of cooperative binding, wherein such cooperative binding is characterized by a cooperativity factor (a) greater than 1 . By supporting the activity of endogenous inhibitory receptor agonists (orthosteric ligands), the modulators potentiate and/or prolong immunosuppressive signaling pathways, offering a targeted and effective approach to managing inflammatory conditions, including autoimmune diseases. In this context, developing therapeutic agents that modulate the cooperativity of inhibitory immune receptor complexes remains largely unexplored, presenting a unique opportunity for innovative research and drug discovery.

The allosteric modulators described herein exhibit specificity for conformational epitopes on immunoinhibitory receptor-ligand complexes. More precisely, these modulators form ternary (or higher-order) assemblies with naturally occurring and transiently bound immunoinhibitory complexes. As a result, their activity is restricted to sites where both receptor and ligand are present simultaneously - for exam pie, atthe immunological synapse where T cell activation occurs. This contrasts with classical immunoinhibitory receptor agonists, which activate receptors systemically wherever the receptor is present, regardless of ligand presence.

Therefore, the modulators of the present invention are advantageous in that they enhance the natural immunosuppressive response locally, providing spatiotemporal selectivity. Consequently, they may offer improved patient tolerability by reducing off-target effects and associated toxicity. The ligand dependency of these positive allosteric modulators may also allow for higher and more frequent dosing compared to conventional drugs, ensuring continuous saturation and improved therapeutic efficacy. Hence, PAMs of this invention will be useful in treatment of inflammatory diseases, including autoimmune diseases or allergies.

In various embodiments, the binders of this invention are ISVD-comprising polypeptides derived from mammalian immune systems, including antibodies or antibody fragments such as Nanobodies®, and their optimized variants and/or formats.

Another aspect of this disclosure pertains to methods for generating the allosteric modulators described above. The method involves a selection process in which candidate binders are chosen based on their ability to: (i) bind to a unique conformation present in an immunoinhibitory complex, and (ii) induce positive cooperativity within such complexes. Additionally, binders may be selected for their capacity to modulate downstream receptor signaling - such as that of PD-1 »PD-L1 , PD- 1 »PD-L2, TIGIT’PVR, CTLA-4»B7.1 , VSTM1 »Gal-1 , LILRB2»Angptl2, or any other immunoinhibitory complex, without limitation. These methods may involve the display of candidate modulators on cell surfaces, such as yeast or mammalian cells.

DESCRIPTION OF THE FIGURES

The drawings provided are schematic and non-limiting. In the drawings, the size of certain elements may be exaggerated or not to scale for illustrative purposes.

Figure 1. Examples of inhibitory immune receptors as possible constituents of protein complexes targeted by the allosteric modulators of this invention.

Figure 2. Examples of ligands of inhibitory immune receptors as possible constituents of protein complexes targeted by the allosteric modulators of this invention.

Figure 3. Examples of immunoinhibitory receptor-ligand complexes as possible targets of the allosteric modulators of this invention.

Figure 4. The cubic ternary complex model of allosteric modulation of receptor activation.

R represents a receptor, for instance hPD-1 , L represents a ligand, for instance hPD-L1. The receptor exists in two conformational states: an inactive state, R, and an active state, R*. The equilibrium between these states is defined by the activation constant K_act. An orthosteric agonist L binds to the inactive receptor R with an equilibrium dissociation constant K_L. An allosteric modulator- such as an allosteric nanobody (Nb) - binds to the inactive receptorwith an equilibrium dissociation constant K_Nb. The model includes four key cooperativity factors: a: the factor of binding cooperativity between the orthosteric agonist L and the allosteric modulator Nb.

0: the factor of cooperativity between the orthosteric agonist binding (L) and receptor activation (R -> R*). y: the factor of cooperativity between the allosteric modulator binding (Nb) and receptor activation (R -> R*).

6: the factor of cooperativity between the allosteric modulator (Nb) and agonist L-induced receptor activation.

Adapted from Jakubik etal., 2020²⁹.

Figure 5.

(A) Strategy to raise allosteric Nanobodies that stabilize immunoinhibitory complexes and act as allosteric modulators. Receptors at rest predominantly adopt an inactive conformation (R). Binding of a ligand, whether a small molecule or a protein, stabilizes the receptor in a signaling- competent active conformation (R*). Immunizing mice with the receptor alone (left) leads to the maturation of conventional monoclonal antibodies (or nanobodies) that bind to the inactive conformation, thereby inhibiting the receptor. Conversely, immunizing llamas with cross-linked receptor-ligand complexes locks the receptor in the ligand-imprinted active-state conformation (right). This approach leads to the maturation of allosteric Nanobodies that stabilize the receptor in its active-state conformation (R*).

(B) On-target allosteric effects of an allosteric Nanobody on the affinity and the efficacy of a receptor-ligand complex, such as the hPD-1»hPD-L1 complex.

Affinity modulators induce a conformational change that alters the kinetics of ligand binding (a). Efficacy modulators induce a conformational change that is transmitted to regions of the receptor involved in signal transduction (6). Allosteric agonists or antagonists induce a response (change in receptor activity), even in the absence of the ligand (y). a, the factor of binding cooperativity between the orthosteric ligand (e.g., hPD-L1 ) and the allosteric nanobody (Nb); 0, the factor of cooperativity between ligand (e.g., hPD-L1 ) binding and receptor (e.g., hPD-1 ) activation; y, the factor of cooperativity between the binding of the allosteric Nb and direct receptor (e.g., hPD-1) activation; 6, the factor of cooperativity between the binding of the Nb and ligand (e.g., hPD-L1)-dependent receptor (e.g., hPD-1 ) activation.

Figure 6. Chemical cross-linking of the hPD-1 »hPD-L1 complex with glutaraldehyde. Samples were incubated with different concentrations of glutaraldehyde, then separated on precast gradient SDS-PAGE gels (Biorad, Mini-protean TGX) and transferred onto a 0,2 pm transblot mini PVDF membrane (Biorad, 1704156). A mouse anti-histidine tag antibody (Biorad, MCA1396) was used in western blotto visualize hPD-1 and hPD-L1 thatwere both His-tagged. The PageRuler™ prestained Protein Ladder (Thermo Scientific, 26616) was used as a molecular weight marker.

Figure 7. Four-step procedure to select allosteric Nanobodies that bind and stabilize the hPD- 1 »hPD-L1 complex. Left: Schematic representations of phage display biopanning Rounds 1a and 2a, where we immobilized hPD-1 and added phages supplemented with an excess of hPD-L1 in solution (top), and Rounds 1 b and 2b, where we immobilized hPD-L1 and used an excess of hPD-1 in solution (bottom). Right: To enrich Nbs that bind to and stabilize the hPD-1 »hPD-L1 complex, we performed 4 consecutive rounds of biopanning where we alternated between hPD-1 and hPD-L1 as the immobilized protomer and the protomer supplemented in solution, respectively.

Figure 8. Representative outcome of a comparative ELISA screen on 96 different periplasmic extracts containing different Nanobodies for binding to the cross-linked hPD-1»hPD-L1 complex or the separate protomers. Different wells were coated with hPD-1 , hPD-L1 or the crosslinked complex of hPD-1 »hPD-L1 to screen for periplasmic extracts containing different Nanobodies selected via 4 rounds of biopanning accordingto Example 4.

Figure 9. Representative outcome of an ELISA screen on 96 different periplasmic extracts containing different Nanobodies for binding to the non-cross-linked hPD-1*hPD-L1 complex. For each Nb, the signal is compared to the signal of a well that was not coated with hPD-1 (control). Well H12 contained an irrelevant Nanobody. Wells A3, A4, A9, B3, B4, C2, C4, C8, C11 , D4, D5, F9, F10, G1 , G2, E2, E9, E11 contained sequence variants of CA18811 (SEQ ID NO: 12). Wells C7 and F11 contained sequence variants of CA19275 (SEQ ID NO: 13). A8, A10, A12, B6, B7, B10, B11 , C3, C5, C9, D3, E7, E8, F2, F3, G4, G5, G6, G11 contained sequence variants of CA19281 (SEQ ID NO: 15).

Figure 10. Association and dissociation isotherms for Nanobodies CA18811 (SEQ ID NO: 12), CA19275 (SEQ ID NO: 13), and CA19281 (SEQ ID NO: 15) upon incubation with the hPD-1«hPD- L1 complex, hPD-1 alone or hPD-L1 alone, measured by Biolayer Interferometry (BLI) on Octet. The K_d values calculated from the association and dissociation isotherms of the hPD-1 »hPD-L1 complex are apparent dissociation constants that reflect properties of ternary complexes, whereas the other measurements concern binary complexes.

Figure 11. Nanobodies that act as allosteric affinity modulators of the hPD-1 •hPD-L1 complex. The biotinylated ectodomain of the human PD-L1 was immobilized on a streptavidin-coated Octet sensor and incubated with increasing amounts of the hPD-1 ectodomain to measure association and dissociation isotherms and obtain binding saturation curves in the presence or absence of saturating amounts of a series of allosteric Nanobodies, a, the factor of binding cooperativity between the orthosteric ligand (hPD-L1) and the allosteric nanobody tested.

Figure 12. Affinity modulation of hPD-L1 binding to hPD-1 expressed on the surface of Jurkat cells by allosteric nanobodies, as analyzed by flow cytometry (FACS). Jurkat cells stably transfected to express an engineered human PD-1 (hPD-1) on the cell surface (Eurofins, DiscoverX) were used. Binding of fluorescently labeled hPD-L1 (hPD-L1 -His-PE, BioTimes, P1009H6-25) to these cells was assessed by fluorescence-activated cell sorting (FACS) in the presence or absence of a candidate positive allosteric modulator.

Figure 13. Crystal structure of CA19279 in a complex with hPD-1. (A) CA19279 (SEQ ID NO: 14) acts as an affinity modulator of the hPD-1 »hPD-L1 complex. (B) Crystal structure of CA19279 (space filling representation) in complex with hPD-1 (ribbon representation in blue). hPD-L1 was modeled based on the structure of the PD-1 »PD-L1 complex (PDB:4ZQK). (C) Intramolecular hydrogen bonds at the interface of CA19279 binding to hPD-1 .

Figure 14. Nanobodies that act as affinity modulators of the hPD-1 •hPD-L1 interaction do not modulate the hPD-1»hPD-L2 complex. Left: The ectodomain of hPD-1 (hPD-1 -Fc) was immobilized on an ELISA plate and incubated with increasing concentrations of biotinylated hPD- L1 (hPD-L1-Fc-Avi) to measure complex formation in the presence of an excess of different Nanobodies or antibodies. Right: The ectodomain of hPD-1 (hPD-1 -Fc) was immobilized on an ELISA plate and incubated with increasing concentrations of biotinylated hPD-L2 (hPD-L2-Fc-Avi) to measure complex formation in the presence of an excess of different Nanobodies or antibodies.

Figure 15. A cell-based reporter assay demonstrating the recruitment of SHP1 to hPD-1. Reporter Jurkat cells overexpressing hPD-1 -ED and hSHP1 -EA (ED and EA: Enzyme Donor and Acceptor domains of a beta-galactosidase, respectively) were incubated with different amounts of hPD-L1 -overexpressing (Raji-PD-L1 ) or PD-L1 -negative (Raji-Null) Raji cells. The resulting luminescence, due to the formed beta-galactosidase, was measured as described in Example 12. The mean and average of two replicates are represented.

Figure 16. Nanobodies that behave as allosteric affinity modulators of the hPD-1»hPD-L1 complex improve the recruitment of SHP1 to hPD-1 in a hPD-L1-dependent manner. Doseresponses of different antibodies on SHP1 recruitment reporter activity in the presence of Raji cells overexpressing hPD-L1 (Raji-PD-L1 ) compared to Raji cells not overexpressing hPD-L1 (Raji-Null). (A) Nanobody 102C12 and pembrolizumab are known antagonists of the hPD-1 pathway. (B) Rosnilimab and peresolimab are known agonists of the PD-1 pathway. (C) CA18811 , CA19281 , and CA19275 are Nanobodies that act as allosteric affinity modulators of the hPD-1 »hPD-L1 complex in biophysical assays (Example 8). The mock Nanobody binds an irrelevant protein. All tests were performed in duplicates; mean and average data, with non-linear regression fit curves, are represented.

Figure 17. EC₅o and E_max of the hPD-1»hPD-L1 complex-stabilizing Nanobodies in the SHP1 recruitment assay in the presence of Raji-PD-L1.

Figure 18. Nanobodies that behave as allosteric affinity modulators of the hPD-1»hPD-L1 complex inhibit activation of NFAT signalling in Jurkat T cells, in a PD-L1-dependent manner.

(A) Jurkat NFAT reporter cells were treated with different conditions: untreated (PBS), IgG (as a control for antibodies), the PD1 antagonist antibody pembrolizumab, the PD1 agonist rosnilimab, an irrelevant Nb, or the PD1 antagonist nanobody 102C12. These treatments were administered in the presence of eitherTHP-1 cells and 0.3 pg/mL anti-CD3 alone or of THP-1 cells, 0.3 pg/mL anti- CD3, and 3 pg/mL PD-LI -Fc.

(B) Jurkat NFAT reporter cells were treated similarly with increasing amounts of CA18811 (SEQ ID NO: 12) or CA19281 (SEQ ID NO: 15), two Nanobodies that act as allosteric affinity modulators of the hPD-1 »hPD-L1 complex in biophysical assays (Example 8). These treatments were administered in the presence of TH P-1 cells and 0.3 pg/mL anti-CD3 alone (left), and with or without 3 pg/mL PD-L1 -Fc (right). All tests were performed in duplicates, and mean values with non-linear regression fit curves are shown, where applicable.

Figure 19. Chemical cross-linking of the hPD-1»hPD-L2 complex with glutaraldehyde. Samples were incubated with different concentrations of glutaraldehyde, then separated on precast gradient SDS-PAGE gels (Biorad, Mini-protean TGX) and stained in InstantBlue protein staining solution (Expedeon, 1SB1 L). The PageRuler™ pre-stained Protein Ladder (Thermo Scientific, 26616) was used as a molecular weight marker. Proteins were transferred to nitrocellulose and the blot was developed with an anti-His antibody that binds the His-tag fused to both hPD-1 and hPD-L2.

Figure 20. Representative outcome of an ELISA screen on 96 different periplasmic extracts containing various Nanobodies. No antibody was added to well H11 , while well H12 was incubated with a periplasmic extract containing an irrelevant Nanobody. Sequencing revealed that wells B2 and H2 contained sequence variants of CA19998 (SEQ ID NO: 242).

Figure 21. Nanobody CA19998 acts as a positive allosteric modulator of the hPD-1»hPD-L2 complex. The biotinylated ectodomain of human PD-1 was immobilized on a streptavidin-coated Octet sensor and incubated with increasing amounts of the hPD-L2 ectodomain to measure the association and dissociation isotherms in the presence or absence of saturating amounts of the allosteric Nanobody CA19998 (SEQ ID NO: 242). Figure 22. Ligand-specific recruitment of SHP1 to hPD-1 by allosteric affinity modulators of hPD-1»hPD-L1 or hPD-1»hPD-L2. Reporter Jurkat cells overexpressing hPD-1-ED and hSHP1 -EA were incubated with Raji cells non-overexpressing PD-L1 (Raji-Null) alone or in the presence of either hPDL-1-Fc or hPD-L2-Fc, and with 5pM of an untargeted Nanobody (mock), or the anti-PD1 antagonist Nanobody (102C12), or the anti-PD-L2 antagonist Nanobody CA19999, or the hPD- 1 »hPD-L1 stabilizing Nanobody CA19275, orthe hPD-1 »hPD-L2 stabilizer CA19998, orwith 100 nM of the PD-1 agonist antibody rosnilimab. Mean and average of 2 experimental replicates are represented.

Figure 23. Nanobodies that act as affinity modulators of the hPD-1 •hPD-LI interaction do not modulate the hPD-1»hPD-L2 complex and vice versa. Left: The ectodomain of hPD-1 (hPD-1 -Fc) was immobilized on an ELISA plate and incubated with increasing concentrations of biotinylated hPD-L2 (hPD-L2-Fc-Avi) to assess complex formation in the presence of an excess of different Nanobodies. Right: The ectodomain of hPD-1 (hPD-1 -Fc) was immobilized on an ELISA plate and incubated with increasing concentrations of biotinylated hPD-L1 (hPD-L1 -Fc-Avi) to measure complex formation in the presence of an excess of the different Nanobodies.

Figure 24. CA19279 amino acid sequence and illustration of the different CDR annotations referred to in this application. CDR annotations according to MacCallum, AbM, Chothia, Kabat and IMGT are shown in colored boxes corresponding to the sequence of Nanobody CA19279 (SEQ ID NO: 14). The CDRs according to Kabat annotation are specified in Table 1 (CDR1 , SEQ ID NO: 17; CDR2, SEQ ID NO: 18; CDR3, SEQ ID NO: 19). For amino acid residue numbering of the sequence, the Kabat numbering system was applied.

Figure 25. CA19281 amino acid sequence and illustration of the different CDR annotations referred to in this application. CDR annotations according to MacCallum, AbM, Chothia, Kabat and IMGT are shown in colored boxes corresponding to the sequence of Nanobody CA19281 (SEQ ID NO: 15). The CDRs according to Kabat annotation are specified in Table 1 (CDR1 , SEQ ID NO: 20; CDR2, SEQ ID NO: 21 ; CDR3, SEQ ID NO: 22). For amino acid residue numbering of the sequence, the Kabat numbering system was applied.

Figure 26. Immunization of a llama with a cross-linked mammalian-produced hPD-1»hPD-L1 complex to generate an antibody immune library.

(A) To covalently stabilize the hPD-1 »hPD-L1 complex, glutaraldehyde was used as a chemical cross-linker in the concentration range from 0.25 % to 0 %. hPD-1 , hPD-L1 or the hPD-1 »hPD-L1 complex was incubated with the respective concentrations of glutaraldehyde at 20°C in a total volume of 15 pl. The reaction was stopped after 30 minutes by the addition of 30 mM Tris-HCl, pH 8.0. The level of cross-linking (CL) was monitored by SDS-PAGE on precast gradient SDS-PAGE gels (Biorad, mini-protean TGX) stained with Instant-Blue Coomassie protein stain (Abeam, ab119211 ), and anti-His western blot using 0.2 pm transblot mini PVDF membrane (Biorad, 1704156) and a mouse anti-Histidine tag antibody (Biorad, MCA1396). As shown, the addition of glutaraldehyde causes the formation of a higher molecular weight band corresponding to the cross-linked hPD- 1 »hPD-L1 complex, which is absent in the self-crosslinked PD-1 or PD-L1 samples where only 1 protein partner of the complex is present.

Figure 27. Validation of molecules acting as allosteric affinity modulators of a post- translationally modified hPD-1»hPD-L1 complex by BLI on the Octet system.

The biotinylated ectodomain of either human PD-1 (for conditions with A00111 and A00123) or human PD-L1 (for conditions with A00118 and the "no VHH" control) was immobilized on a streptavidin-coated Octet sensor and incubated with increasing concentrations of the corresponding hPD-L1 or hPD-1 ectodomain in solution, respectively, to measure association and dissociation isotherms and generate binding saturation curves. These measurements were performed in the presence or absence of saturating concentrations of the respective VHH (allosteric modulator). The resulting apparent K_d values and a factors are indicated in the table.

Figure 28. Sequence alignment.

Framework alignment of Nanobody sequences to the human IGHV3-JH germline sequence, exemplifying a sequence humanization and optimization process using the IMGT numbering system. The parental sequence CA19281 (SEQ ID NO: 15) is aligned with the framework-optimized variant A00032 (SEQ ID NO: 49), single-mutation variants A00043 (SEQ ID NO: 51) and A00044 (SEQ ID NO: 50), and multiple-mutation variants A00235 (SEQ ID NO: 53), A00236 (SEQ ID NO: 58), A00241 (SEQ ID NO: 56), and A00242 (SEQ ID NO: 57). CDR1 , CDR2, and CDR3 regions are shaded in light gray; VHH hallmark residues are highlighted in dark gray. Dots indicate amino acid residues identical to the reference sequence, and potential post-translational modification motifs within the CDRs are shown in bold and underlined.

Figure 29. Sequence-optimized and humanized Nanobodies derived from the parental CA19281 maintain comparable activity in the SHP1 recruitment assay.

(A) Dose-response comparison of the parental CA19281 (SEQ ID NO: 15) molecule with the framework-optimized variant A00032 (SEQ ID NO: 49). (B) Dose-response comparison of the framework-optimized variant A00032, and subsequent single mutants A00043 (SEQ ID NO: 51) and A00044 (SEQ ID NO: 50). (C) Dose-response of the framework-optimized variant A00032 (SEQ IDNO: 49) compared to fully optimized variants A00235 (SEQ ID NO: 53), A00236 (SEQ ID NO: 58), A00241 (SEQ ID NO: 56), and A00242 (SEQ ID NO: 57). (D) Dose-response comparison of the parental CA18810 (SEQ ID NO: 11 ) molecule with the framework-optimized variant A00255 (SEQ ID NO: 54) and fully sequence-optimized variant A00343 (SEQ ID NO: 55). All graphs represent the mean of two replicates, with non-linear regression curves fitted to the data.

Figure 30. Formatting Nanobodies for half-life extension does not impair - and may even enhance - potency in the SHP1 recruitment assay in a ligand-dependent manner.

(A) Left panel: fusion of the monovalent Nanobody CA19281 (as its framework-optimized variant, A00032) to an anti-serum albumin Nanobody (resulting in A00051 ) does not affect the SHP1 recruitment potency compared that of the parent. Middle panel: increasing valency by repeating the CA19281 Nanobody in bi- (A00055), tri-, or tetravalent (A00278) formats enhances pharmacological potency. Right panel: fusing CA19281 and CA19275 with an anti-serum albumin Nanobody (resulting in A00137) improves SHP1 recruitment potency compared to either parent alone.

(B) Variations in linker length, including (GGGGS)1 (SEQ ID NO: 64) in A00144, (GGGGS)2 (SEQ ID NO: 66) in A00147, (GGGGS)4 (SEQ ID NO: 68) in A00150, and (GGGGS)7 (SEQ ID NO: 59) in A00055, result in comparable EC₅₀ values in the SHP1 recruitment assay.

(C) Fusion of the monovalent nanobody CA19281 (as its framework-optimized variant, A00032) to an Fc tail, resulting in A00062 (wild-type Fc) or A00065 (LALAPG (SEQ ID NO: 71 )-mutated Fc), enhances SHP1 recruitment potency.

(D) Despite bivalent formatting, both the serum-albumin-VHH-fused CA19281 (A00055, left panel) and the IgG-fused variant (A00062, right panel) retain strict PD-L1-dependent activity. This is evidenced by the lack of activity observed in Raji-Null cells, which do not express PD-L1 , confirming that the functional effect of these constructs requires ligand engagement.

(E) The bivalent format with serum-albumin-VHH -fusion of A00255 (resulting in A00265) improves SHP1 recruitment potency compared to that of the parent molecule A00255.

Figure 31. Comparison of association and dissociation kinetics of the PD-1»PD-L1 complex in the presence of monovalent A00032 or bivalent A00055 or A00062, by BLI.

The bivalent molecules A00055 and A00062 exhibited a marked improvement in apparent dissociation rate constant (k_off ^app) compared to the monovalent molecule A00032, as evidenced by their slower dissociation beginning at approximately 4000 seconds.

Figure 32. NFAT signaling of formatted hPD-1»hPD-L1 PAMs. (A) The bivalent format with serum-albumin-VHH -fusion of A00255 (resulting in A00265) improves NFAT signaling potency and efficacy compared to framework-optimized parent A00255. (B) The bi- and trivalent formats with serum-albumin-VHH -fusion of CA19281 (resulting in A00055 and in A00153, respectively) improve NFAT signaling potency compared to monovalent serum-albumin- VHH -fusion A0051 . (C) The IgGI fusion of CA19281 (resulting in A00062) improves NFAT signaling potency compared to parent and compared to its irrelevant lgG1 fusion Nb control. Plots represent average of 4 (A and B) and 2 (C) replicates with standard deviation and non-linear regression fits.

Figure 33. Cytokine release in a mixed lymphocyte reaction (MLR).

Levels of IFNy and TNFa released into the supernatant were measured 5 days after co-culturing PBMCs from two healthy donors to trigger a mixed lymphocyte reaction (MLR) and T cell activation. Treatments included vehicle (Baseline MLR), 160 nM of an irrelevant IgGFc-fused Nanobody, the hPD-1 »hPD-L1 IgGFc-fused PAM A00062 (SEQ ID NO: 70), or 10 nM of the PD-1 agonist monoclonal antibodies peresolimab and rosnilimab. Cytokine concentrations are presented as fold-change relative to the vehicle condition. Each symbol reflects the geometric mean of triplicate measurements from a given donor pair, while the line represents the geometric mean across all donor pairs. Asterisks (*) denote statistical significance (p < 0.05) versus vehicle based on a two- way ANOVA (factors: donor pair and treatment, applied to raw cytokine data), while "ns" indicates comparisons that were not statistically significant.

Figure 34. Cytokine release in a mixed lymphocyte reaction (MLR).

Levels of IFNy and TNFa released into the supernatant were measured 5 days after co-culturing PBMCs from two healthy donors to trigger a mixed lymphocyte reaction (MLR) and T cell activation. Treatments included vehicle (Baseline MLR), 160 nM of an irrelevant IgG-fused Nanobody, the hPD- 1 »hPD-L1 IgG-fused PAM A00062 (SEQ ID NO: 70), or 10 nM of the PD-1 agonist monoclonal antibodies peresolimab and rosnilimab. Cytokine concentrations are presented as fold-change relative to the vehicle condition. Each symbol reflects the geometric mean of triplicate measurements from a given donor pair, while the line represents the geometric mean across all donor pairs. Asterisks (*) denote statistical significance (p < 0.05) versus vehicle based on a two- way ANOVA (factors: donor pair and treatment, applied to raw cytokine data), while "ns" indicates comparisons that were not statistically significant.

Figure 35. Characterization of epitopes via epitope binning using BLI.

To determine whether the identified PD-1 »PD-L1 PAMs bind to overlapping or non-overlapping epitopes, an epitope binning experiment was performed employing BLI on an OCTET R8 instrument. Biotinylated CA18811 (panel A), CA19275 (panel B), CA19279 (panel C) or CA19281 (panel D) were immobilized on SA-coated sensors and plunged into a solution containing 1 pM hPD-1 »hPD-L1 complex in the presence or absence of 5 pM PAM. Sensorgrams (in nanometers, nm) were compared against both the negative control (the same VHH immobilized and in solution) and the positive control (no VHH). If the PAM VHH in solution binds to an overlapping epitope, competition occurs, resulting in a flat response (comparable to the self-competition negative control). Signals higher than that - and possibly even higher than the positive control (no VHH) - indicate that the immobilized VHH can bind the complex in the presence of the PAM in solution, revealing the existence of a second, non-overlapping epitope. The binding profiles and epitope overlaps are summarized in panel (E). It can be concluded that CA18811 , CA19275 and CA19281 bind to an overlapping (largely similar) epitope A, while CA19279 binds to a distinct (non-overlapping) epitope B. Each PAM was tested in both orientations, providing two independent determinations.

Figure 36. Deep sequencing analysis of sequence-optimized variants A00247-A00253, derived from the same clonal cluster.

The sequence diversity within the complementarity-determining regions (CDRs), as defined by the IMGT numbering scheme, is illustrated for these related variants. Diversity is shown as sequence logo plots and as numerical frequency per amino acid (aa) position for (A) CDR1 , (B) CDR2, and (C) CDR3.

Figure 37. Sequence alignment and CDR similarity analysis of hPD-1»hPD-L1 PAMs of the same clonal cluster/family.

Amino acid sequences and their alignment of molecules (SEQ ID NO: 35), A001 13 (SEQ ID NO: 36), A00114 (SEQ ID NO: 37), A001 15 (SEQ ID NO: 38), A00116 (SEQ IDNO: 39), A001 17 (SEQ ID NO: 40), A001 18 (SEQ ID NO: 41 ), A00119 (SEQ ID NO: 42), A00120 (SEQ ID NO: 43), A00121 (SEQ ID NO: 44), A00122 (SEQ ID NO: 45), A00123 (SEQ ID NO: 46), A00124 (SEQ ID NO: 47) and A00183 (SEQ ID NO: 48).

Numbering based on IMGT, with CDR1 , -2, and -3 highlighted in lighter gray ; VHH hallmark residues are highlighted in dark gray. Dots indicate amino acid residues identical to the reference sequence, and potential post-translational modification motifs within the CDRs are shown in bold and underlined.

Figure 38. Chemical cross-linking of the hCTLA-4»hB7.1 complex with glutaraldehyde.

Samples of the hCTLA-4»hB7.1 complex, either untreated (noCL) or cross-linked with 0.05% glutaraldehyde (CL), were separated on precast gradient SDS-PAGE gels (Bio-Rad, Mini-PROTEAN TGX), followed by transfer onto 0.2 pm Trans-Blot Mini PVDF membranes (Bio-Rad, 1704156). Detectionwas performed via Western blot usinga mouse anti-Histagantibody (Bio-Rad, MCA1396) to visualize both His-tagged components of the complex. The PageRuler™ Pre-stained Protein Ladder (Thermo Scientific, 26616) served as the molecular weight marker.

Figure 39. Two-step procedure to select allosteric Nanobodies that bind and stabilize the hCTLA-4»hB7.1 complex.

Left: Schematic representations of phage display biopanning Round 1 a (top), in which hCTLA-4was immobilized and phage libraries were incubated in the presence of excess soluble hB7.1 , and Round 1 b (bottom), where hB7.1 was immobilized and excess soluble hCTLA-4 was added.

Right: To enrich for nanobodies that specifically recognize and stabilize the hCTLA-4»hB7.1 complex, two consecutive rounds of biopanning were conducted. In each round, the immobilized protomer and the soluble counterpart were alternated between hCTLA-4 and hB7.1 , respectively.

Figure 40. Representative results from the FACS-based screen of 96 distinct periplasmic extracts, each containing a unique Nanobody.

(A) Schematic representation of Raji B cells stained with hCTLA-4-Fc.

(B) FACS-based screening results. For each nanobody, the fold change in signal was calculated by dividing the mean fluorescence intensity (MFI) by the average MFI of six negative control wells containing an irrelevant Nb (wells H3, H5, H6, H7, H8, H10). Dashed lines indicate the mean ± 3 times the standard deviation (SD) of all controls. Wells A01 , A06, B04, B09, C05, C06, C12, D09, E08, F03, F08, G02, and G03 contained sequence variants of A00176 (SEQ ID NO: 246) . Wells A02, A03, A04, A05, A07, A09, A11 , A12, B01 , B02, B03, B05, B07, B08, B10, B12, C01 , C10, D03, D04, D05, D07, D08, D11 , D12, E02, E04, E07, E11 , F01 , F02, F05, F06, F09, G01 , G04, G05, G06, G09, G10, and G12 contained sequence variants of A00175 (SEQ ID NO: 245). Well D10 contained a sequence variant of A00179 (SEQ ID NO: 247)

Figure 41. Nanobodies that act as allosteric affinity modulators of the hCTLA-4»hB7.1 complex.

The biotinylated ectodomain of hCTLA-4 was immobilized on a streptavidin-coated Octet sensor and incubated with increasing concentrations of the hB7.1 ectodomain to measure association and dissociation isotherms. Binding saturation curves were generated in the presence or absence of saturating concentrations of a series of allosteric nanobodies: A00175 (SEQ ID NO: 245), A00176 (SEQ ID NO: 246), A00179 (SEQ ID NO: 247), and A00187 (SEQ ID NO: 248). Figure 42. Affinity modulation of the hCTLA-4»hB7.1 interaction at the surface of Raji B cells by allosteric nanobodies, as assessed by flow cytometry (FACS).

The fluorescence of each sample of cells was monitored by calculating the mean fluorescence intensity (MFI) of ± 2x10⁴ cells.

Figure 43. Association and dissociation isotherms for Nbs A00187 upon incubation with the protein complexes or individual protomers, as measured by BLI.

Interactions were measured at a protein concentration of 300 nM for the following targets: hCTLA- 4»hB7.1 , hCTLA-4, hB7.1 , hCTLA-4»hB7.2, hCD28»hB7.1 , and hCD28»hB7.2

Figure 44. Nanobodies that behave as allosteric affinity modulators of the hCTLA-4»hB7.1 complex dampen the TCR/CD28 signaling.

Luminescence signal from the TCR/CD28 activation reporter Ju rkat cell line overexpressing hCTLA- 4 was measured upon co-culture with APC-like cells expressing B7.1 and B7.2, in the presence of varying concentrations of ipilimumab, abatacept, an irrelevant Nanobody, or the CTLA-4»hB7.1 complex-stabilizing Nanobodies A00175 and A00179. Data are presented as the mean ± standard deviation of two replicates. Non-linear regression curves were fitted to the dose-response data.

Figure 45. Nanobodies that are allosteric affinity modulators of the hCTLA-4»hB7.1 complex reduce B7.1 -induced T cell activation.

Percentage of CD25-positive human isolated T cells cultured for 72 hours with control beads (coated with lysozyme) or activating beads (coated with anti-CD3), either alone or in the presence of anti-CD28 antibody or B7.1 -Fc. Treatments included vehicle only (PBS), irrelevant Nanobody, CTLA-4»hB7.1 -complex stabilizing Nanobody A00175 (at 3000, 600, and 120 nM), or abatacept (at 100 and 20 nM). The plot shows the mean and standard deviation of two replicates. An asterisk (*) indicates a statistically significant difference compared to the vehicle control in the anti-CD3 plus B7.1-Fc condition (p < 0.05, one-way ANOVA), while “ns” denotes no significant difference.

Figure 46. Immunization of a llama with a cross-linked hCD200R»hCD200 complex to generate an antibody immune library.

To covalently stabilize the hCD200R»hCD200 complex, glutaraldehyde was used as a chemical cross-linker in the concentration range from 0.3 % to 0 %. hCD200R, hCD200 or the hCD200R»hCD200 complex was incubated with the respective concentrations of glutaraldehyde at 4 °C in a total volume of 20 pl. The reaction was stopped after 30 minutes by the addition of 50 mM Tris-HCl, pH 7.4. The level of cross-linking was monitored by SDS-PAGE on precast gradient SDS-PAGE gels (Biorad, mini-protean TGX) stained with Instant-Blue Coomassie protein stain (Abeam, ab1 1921 1 ), and anti-His western blot using 0.2 pm transblot mini PVDF membrane (Biorad, 1704156) and a mouse anti-Histidine tag antibody (Biorad, MCA1396). As shown, the addition of glutaraldehyde causes the formation of a higher molecular weight band corresponding to the cross-linked hCD200R»hCD200 complex.

Figure 47. Two-step selection strategy for identifying allosteric Nanobodies that bind and stabilize the hCD200R»hCD200 complex.

Left: Schematic overview of phage display biopanning. In Round 1 a, biotinylated hCD200R was immobilized, and phages were incubated in the presence of excess soluble hCD200 to counterselect binders to hCD200 alone. In Round 1 b, the setup was reversed: hCD200 was immobilized while excess soluble hCD200R was used in solution. By alternating the immobilized and soluble protomer across the two rounds, this strategy enriches for Nanobodies that recognize epitopes specific to the hCD200R»hCD200 complex.

Right: The corresponding inverse setup, in which hCD200 was immobilized in Round 1 a and hCD200R in Round 1 b, was performed in parallel.

Figure 48. Screening for Nanobodies that stabilize the non-cross-linked hCD200R*hCD200 complex by ELISA.

Fold change of signal for each Nanobody Nb was calculated by dividing the optical density (OD) of the Nb by the OD of the reference containing an irrelevant VHH. Error bars represent standard deviations (SD). An increase in signal compared to the irrelevant VHH indicates that the Nanobody stabilizes the interaction between hCD200R and hCD200, while a decrease indicates inhibition of this interaction. Nanobodies A00267, A00268, A00269, A00270, A00271 , A00274, A00276, and A00277 are thus identified as candidate positive allosteric modulators (PAMs) of the hCD200R»hCD200 complex.

Figure 49. Characterization of purified Nanobodies for binding to hCD200R, hCD200, or the cross-linked hCD200R»hCD200 complex by ELISA.

For each Nb, the fold change in signal was determined by dividing its optical density (OD) by that of the corresponding irrelevant Nb reference. A dashed line marks the threshold of twice the reference value; clones exceeding this were considered binders to the coated antigen. Based on this criterion, A00267 (SEQ ID NO: 253), A00268 (SEQ ID NO: 254), A00269 (SEQ ID NO: 255), A00270 (SEQ ID NO: 256), and A00271 (SEQ ID NO: 257) bind both hCD200R-His and the hCD200R»hCD200 complex; A00272 (SEQ ID NO: 261 ) and A00273 (SEQ ID NO: 262) bind hCD200-His and the complex; while A00274 (SEQ ID NO: 258), A00276 (SEQ ID NO: 259), and A00277 (SEQ ID NO: 260) bind exclusively to the crosslinked (CL) hCD200R»hCD200 complex and not to the individual protomers.

Figure 50. BLI of Nanobodiesthat act as allosteric affinity modulators of the hCD200R»hCD200 complex.

The biotinylated ectodomain of human CD200R was immobilized on a streptavidin-coated Octet sensor and incubated with increasing amounts of the hCD200 ectodomain to measure association and dissociation isotherms and obtain binding saturation curves in the presence or absence of saturating amounts of a series of allosteric Nanobodies.

Figure 51. Validation of molecules exhibiting a positive allosteric modulator profile for the hCD200R«hCD200 complex by ELISA.

Dose-response curves obtained from a CD200R titration on ELISA plates in the presence of either allosteric or irrelevant VHH were used to calculate EC₅₀ values. The ratio of the EC₅₀ of the allosteric VHH to that of the irrelevant VHH serves as an estimate of the cooperativity factor a.

Figure 52 . Chemical cross-linking of the hTIGIT»hPVR complex with glutaraldehyde.

Samples of hTIGIT, hPVR, mixed hTIGIT + hPVR and crosslinked (CL) hTIGIT»hPVR complex without or with 0.1 % glutaraldehyde were separated on precast gradient SDS-PAGE gels (Biorad, Mini- protean TGX) then transferred onto a 0.2 pm transblot mini PVDF membrane (Biorad, 1704156). The western blot was developed using a mouse anti-Histidine tag antibody (Biorad, MCA1396) to visualize hTIGIT and hPVR that were both His-tagged. The PageRuler™ pre-stained Protein Ladder (Thermo Scientific, 26616) was used as a molecular weight marker (MWM).

Figure 53. Two-step selection strategy for identifying allosteric Nanobodies that bind and stabilize the hTIGIT»hPVR complex.

Left: Schematic overview of phage display biopanning. In Round 1 a, biotinylated hTIGIT was immobilized, and phages were incubated in the presence of excess soluble hPVR to counter-select binders to hPVR alone. In Round 1 b, the setup was reversed: hPVR was immobilized while excess soluble hTIGIT was used in solution. By alternating the immobilized and soluble protomer across the two rounds, this strategy enriches for Nanobodies that recognize epitopes specific to the hTIGIT’hPVR complex.

Right: The corresponding inverse setup, in which hPVR was immobilized in Round 1 a and hTIGIT in Round 1 b, was performed in parallel. Figure 54. Representative outcome of an ELISA-based screen on 84 different periplasmic extracts, each containing a unique Nanobody.

For each Nanobody, the fold change in signal was calculated by dividing the optical density (OD) of the tested VHH by the average OD of the reference well containing an irrelevant Nb serving as reference. Dashed lines represent the mean ± 3 times the standard deviation (SD) of the reference. Wells A2, A3, A4, A7, A8, B10, C2, C4, C5, C10, D10, E9, F5, F12, G2, and G12 showed values above the +3 SD threshold and were thus considered to contain candidate positive allosteric modulators (PAMs) selected for further analysis, as described hereafter.

Figure 55. Characterization of purified VHHs binding to hTIGIT, hPVR, or the cross-linked hTIGIT-hPVR complex by ELISA

The fold change in ELISA signal (optical density, OD) was calculated by dividing the OD in the presence of a test VHH by the OD obtained with an irrelevant control VHH which served as reference. A dashed line indicates the threshold of a two-fold increase over the reference; clones exceeding this threshold were considered binders to the coated antigen. As a representative example, clone A00314 (SEQ ID NO: 264) binds both hTIGIT-hFc and the hTIGIT*hPVR complex, whereas clone A00312 (SEQ ID NO: 263) binds exclusively to the hTIGIT* hPVR complex and not to the individual protomers.

Figure 56 . Validation of selected VHHs exhibiting a positive allosteric modulator (PAM) profile toward the hTIGIT»hPVR complex by ELISA.

Dose-response curves obtained from hTIGIT titration for hPVR binding in ELISA, in the presence of allosteric or irrelevant VHHs, were used to calculate EC₅o values. The ratio of EC₅o values between conditions with allosteric versus irrelevant VHHs provides an estimate of the cooperativity factor a.

Figure 57. Epitope mapping of the hPD-1*hPD-L1 complex PAM Nbs through screening of a PD- 1 mutant library.

(A) Level of hPD-1 binding to immobilized Nb CA18811 , CA19275 (epitope A binders) or CA19279 (epitope B binder) in the presence of hPD-L1 , as quantified by ELISA. In the left panel, raw signal of the PD-1 WT binding to each of the 3 Nbs show that CA19279 retains more hPD-1 WT than the 2 others at basal level (data points show individual repeats, bars show mean with standard deviations). On the right panel, bars show the relative difference (signal with blanks subtracted and normalized to the level of hPD-1 WT binding per each Nb) for hPD-1 WT binding and for the hPD-1 mutant protein binding for which a difference pertaining to the epitope binding difference was observed comparing the 3 different immobilized Nbs. While E141 K and V43I led to a reduction in signal for CA19279 compared to CA18811 and CA19275, it was the opposite for E61 K and S62Kwith CA19279 having higher binding strength.

(B) Illustrative picture of the epitope regions on hPD-1 . The structure of hPD-1 in complex with hPD- L1 (PDB: 4zqk) is aligned with the structure of hPD-1 in complex with CA19279 (of this disclosure). hPD-L1 in middle grey on top, hPD-1 in light grey in the middle and CA19279 in dark grey at the bottom. Epitope regions are circled around the residues (dark colored) disturbing the binding: mutations in residues E61 , S62 disturb the binding for CA19275 and CA18811 , while mutations in residues V43 and E141 disturb the binding for CA19279.

DESCRIPTION

The present invention is described with reference to specific embodiments and accompanying drawings. However, the invention is not limited to these embodiments or drawings and is defined solely by the appended claims. Any reference signs in the claims are included for clarity only and shall not be construed as limiting the scope of the claims. The invention encompasses all alternatives, modifications, and equivalents that fall within the scope of the claims. It should be understood that not all features or advantages described herein are necessarily achieved in every embodiment. For example, a person skilled in the art will recognize that certain embodiments may achieve or optimize particular advantages, while not necessarily achieving others.

The structure and method of operation of the invention, along with its features and benefits, will be best understood from the following detailed description, read in conjunction with the accompanying drawings. The aspects and advantages of the invention will become apparent from the definitions and embodiments described below. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic is included in at least one embodiment of the invention. The phrases "in one embodiment" or "in an embodiment", appearing in various locations throughout this disclosure, do not necessarily refer to the same embodiment, although they may refer to the same embodiment.

Definitions

As used herein, an indefinite or definite article (e.g., "a", "an" or "the") preceding a singular noun is intended to include the plural form of that noun unless explicitly stated otherwise. The term "comprising", as used in the description and claims, is intended to be inclusive and open-ended. It specifies the presence of the stated features, elements, steps, or components, but does not preclude the presence or addition of one or more other features, elements, steps, or components not explicitly recited. Where the terms "essentially consisting of", "consisting essentially of", or "comprisingsubstantially" are used herein in relation to chemical matter, such as a protein or other compound, they are intended to mean that additional components may be present, provided these do not significantly affect the essential characteristics of the chemical matter. Hence, these terms indicate that the majority or bulk of the chemical matter - such as a polypeptide - defines the functional outcome, although additional elements (e.g., extra amino acids) may be included without altering the core function. Furthermore, terms such as "first", "second", and "third", when used to distinguish steps or elements, do not imply a specific order or sequence, unless explicitly stated. These terms are used merely for differentiation and may be interchanged as appropriate. It follows that the embodiments described herein may be implemented in an order different from that illustrated or described, without departing from the scope of the invention. The terminology employed herein is intended solely to facilitate understanding of the invention. Unless explicitly defined otherwise, all terms shall be interpreted as understood by a person of ordinary skill in the art (e.g., in cell biology, molecular biology, biochemistry, genetics, immunology, structural biology, protein engineering, pharmacology, pharmaceutics, and/or computational biology). For clarity on domain-specific terms and technical usage and/or conventions, please refer to the following sources: i) "Molecular Biology", 3^rd edition (2018), by Clark DP, Pazdernik NJ and McGehee MR³⁰; ii) "Molecular Cloning: A Laboratory Manual", 4^th edition (2012), by Green MR and Sambrook J³¹; iii) "Advances in Protein Molecular and Structural Biology Methods", 1^st edition (2022), byTripathiT and Dubey VK³²; iv) "Fundamentals of Molecular Structural Biology", 1^st edition (2020), by Subrata P³³; and v) "Cellular and Molecular Immunology", 10^th edition (2021 ), by Abbas AK, Lichtman AH and Pillai S³⁴.

In describing and claiming the embodiments of the invention, the following terminology is used.

To comprise

In the context of this invention, the terms "comprising" (and its variations, such as "comprise" or "comprises"), "having" (and its variations, such as "have" and "has"), "including" (and its variations, such as "includes" and "include"), or "containing" (and its variations, such as "contains" or "contain"), are inclusive and open-ended. Therefore, they may encompass additional elements, features and/or method steps which may not be explicitly listed in this application.

Modulator, binding agent

In the context of the present invention, the terms “modulator”, “binding agent” or “binder” can be used interchangeably. These terms refer to a substance, compound, molecule, chemical species or biological particle that, preferably upon specifically binding to a target molecule or molecular complex, as defined herewith, affects, alters or changes: i) the physical state or conformation of said target molecule or molecular complex, and/or ii) the function, activity or reactivity of said target molecule or molecular complex, and/or iii) the quantity or expression level of said target molecule or the constituents/subunits of said molecular complex, and/or iv) the localization of the target molecule or molecular complex. These changes are assessed relative to the absence of the modulator/binding agent/binder or compared to a vehicle or non-binding substitute control. For instance, a modulator may bind to a target protein-protein complex, inducing a conformational change within the complex that increases or decreases the association or dissociation rate of its subunits/constituents, and/or modulates the strength and/or duration of downstream complex signaling. Preferably, the modulator of this invention is an “allosteric modulator”, as subsequently defined.

A modulator/binding agent/binder may be selected from the group comprising, without any limitation, a polypeptide, a peptide, a small molecule (defined as any organic compound with a low molecular weight, e.g., <900 Da or <500 Da), a natural compound, a peptidomimetic, a nucleic acid, a lipid, a lipopeptide, a carbohydrate, an antibody or any fragment derived thereof, such as a fragment antigen-binding (Fab), a disulfide-bonded fragment antigen-binding (F(ab')2), an Fd fragment, a single-chain variable fragment (scFv), a single-chain antibody, a disulfide-stabilized Fv (dsFv), a diabody, and antibody fragments comprising either a VL or VH domain, including a heavy chain antibody (hcAb), a single domain antibody (sdAb), a minibody, a variable domain derived from camelid heavy chain antibodies (VHH or Nanobody™), and a variable domain of the new antigen receptors derived from shark antibodies (VNAR). Furthermore, a modulator/binding agent/binder may be an antibody mimetic, including affibody molecules, affilins, affimers (adhirons), affitins, alphabodies, anticalins, avimers, designed ankyrin repeat proteins (DARPins), fynomers, gastrobodies, Kunitz domain peptides, monobodies, nanoCLAMPs (clostridal antibody mimetic proteins), optimers, repebodies, pronectins, centyrins, and obodies. A modulator/binding agent/binder may also be selected from various protein scaffolds, such as protein A, protein G, fibronectin type III repeats, inhibitor cysteine knots (knottins), and engineered CH2 domains (nanoantibodies).

A target molecule or molecular complex, to which a modulator binds, may encompass a broad range of molecules including amino acids, peptides, proteins, nucleotides, nucleic acids, lipids, carbohydrates, ions, as well as their complexes, aggregates, chains, and/or fragments.

Vehicle Herein, the term “vehicle” refers to an inert medium, substance, or formulation utilized to deliver the binder/modulator under investigation in an experimental setting. The vehicle also serves as a baseline or negative control. In various binding assays, employing the vehicle alone - without the active binder/modulator - allows differentiation between the intrinsic effects of the delivery medium and the specific binding properties of the binder/modulator of interest.

Non-binding substitute control

The term "non-binding substitute control", also referred to herein as “irrelevant control”, refers to an experimental condition employed in binding assays to evaluate the specificity of a modulator or binder for its target. In this control, the target is replaced with a structurally similar molecule that does not bind the modulator under the same assay conditions. Specificity is assessed by comparing the binding signal or affinity observed with the target versus the non-binding substitute.

Allosteric modulator

Pertaining to this invention, the term “allosteric modulator”, “allosteric binding agent”, “allosteric binder”, “allosteric agent” or “allosteric substrate” refers to a binding agent/modulator, as defined herein, that i) binds to a specific site, epitope, or pocket on a target molecule or molecular complex, which is distinct from its orthosteric site(s), and ii) affects the physical state, function, activity, and/or expression level/quantity of the target molecule or molecular complex.

In this context, an “orthosteric site” or an “active site” is understood as the primary binding site of a molecule responsible for mediating its normal, physiological or most common biological function, such as a ligand-binding site of a receptor or a catalytic site of an enzyme. The orthosteric site is generally responsible for the native biological activity of the protein upon ligand engagement and is distinct from allosteric sites, which modulate protein function through binding at topologically separate regions. Thus, an “allosteric site”, “allosteric pocket”, or “allosteric epitope”, also referred to as a “regulatory site”, “regulatory pocket”, or “regulatory epitope”, denotes a specific non-catalytic binding site on a molecule (such as a receptor), distinct from the active/orthosteric site.

Allosteric interactions of proteins play a pivotal role in various biological processes, including enzymatic activity, signal transduction, transcriptional regulation and cell adhesion. For example, an allosteric modulator can induce various effects on a protein receptor, which include: (i) alteration of the receptor's affinity for a molecule (like a natural/endogenous agonistic ligand) binding at a distinct site or epitope (such as an orthosteric site), which can be measured by the cooperativity factor a, as defined herewith; and/or (ii) modulation of receptor signaling efficacy, wherein the receptor signaling efficacy is defined as the ability of an agonistic ligand to activate receptor signaling, possibly measured by the cooperativity factor 6 (see Figure 5B); and/or (iii) direct receptor stimulation that triggers downstream signaling, an effect possibly quantified by the allosteric factor y (see Figure 5B). The direction and magnitude of these effects are not restricted for any given receptor;

Allosteric modulation of proteins, also known as “protein allostery”, can be further elucidated through thermodynamic models that correlate changes in protein conformation with alterations in protein functionality. This perspective has underpinned the conventional theories of allostery, exemplified by the Monod-Wyman-Changeaux (MWC) model³⁵, where the binding of allosteric modulators induces a coordinated conformational transition of the protein subunits toward the active state, and the Koshland-Nemethy-Filmer (KNF) model³⁶, which postulates that the binding of a positive allosteric modulator to a protein component propels it towards the active state, whereby the transition to full activation occurs in a sequential manner. More recently, the ensemble allosteric model (EAM)³⁷ was introduced, offering a framework that interprets allosteric events based on the influence of allosteric binding agents or substrates on the entire conformational spectrum of a protein. Additionally, there is a growing recognition of the significance of dynamics during allosteric interactions^{38 39}, encompassing the role of entropy in energy landscape modeling, which has spurred the development of protein switches⁴⁰. When an allosteric modulator binds to a protein, it alters the free energy landscape within the conformational space of that protein. In this scenario, such alteration impacts not only the protein's thermodynamic properties but also its dynamic features, including the amplitude of motions across various time scales (encompassing motions from the vibration of a single bond to the collective movement of whole domains) and the rates of conformational transitions^{39 41}.

Allosteric binding sites or epitopes, unlike orthosteric binding sites, are subject to less evolutionary pressure, resulting in less conserved structures. This structural variability can provide higher binding selectivity for allosteric binders compared to orthosteric binders⁴². Interestingly, evolutionary adaptation mechanisms can regulate and adjust how allosteric binders influence the proteins they interact with, thereby enhancing their selectivity through optimized “cooperativity” with orthosteric substrates, as defined below⁴³.

It is noteworthy that an allosteric modulator, as described and defined herein, may exhibit selectivity either for a single molecular entity or for a multi-component assembly such as a complex, aggregate, or molecular chain. As a non-limiting example, an allosteric modulator targeting the PD-1 »PD-L1 complex, as disclosed in this application, may bind to one or more conformation-induced allosteric epitope(s) that emerge upon complex formation. These epitopes may include specific amino acid residues located on: i) PD-1 alone, ii) PD-L1 alone, or iii) both PD- 1 and PD-L1 simultaneously. Through such selective binding, the allosteric modulator may influence the cooperative behavior of the complex, thereby modulating downstream signaling events associated with the inhibitory immune receptor pathway.

A successful allosteric modulator binds to an allosteric site and remotely alters the conformation of the primary orthosteric binding site of the biological target. This conformational change can influence the binding of natural ligands to the orthosteric site in one of two principal ways: it may enhance the binding affinity of the orthosteric ligand, resulting in amplified signaling or increased receptor activity - an effect attributed to compounds known as positive allosteric modulators (PAMs); or it may inhibit ligand binding, leading to reduced signaling or receptor activity, characteristic of negative allosteric modulators (NAMs)⁴⁴. The term “positive allosteric modulator” (PAM), as used herein, refers to a ligand that binds to an allosteric site on a receptor or receptorligand complex and enhances the receptor’s response to the orthosteric ligand without independently activating the receptor. A PAM may increase the binding affinity of the orthosteric ligand (positive binding cooperativity), enhance the efficacy of receptor activation once the orthosteric ligand is bound (positive efficacy cooperativity), or exert both effects. PAMs achieve this by stabilizing receptor conformations that are more favorable for orthosteric ligand binding and/or downstream signaling. Their modulatory effect is typically saturable and dependent on the presence of the orthosteric ligand. In immunological contexts, PAMs can amplify receptor- mediated signaling cascades associated with immunosuppression and may be therapeutically exploited to potentiate immunosuppressive responses in a ligand-dependent manner.

Cooperativity, cooperative binding

“Cooperativity”, also known as “cooperative ligand binding” or “cooperative binding”, refers to the phenomenon where the binding of an agent at one site on a molecule/molecular complex, such as a protein or protein complex, influences the binding of another ligand molecule at a different site on the same molecule/molecular complex. For molecules with two or more binding sites, cooperativity is characterized by a change in the intrinsic (site-specific) equilibrium dissociation constant (K_d), as defined herewith, throughout the progression of the binding reactions. This means that the affinity of a given binding site for a substrate is influenced, favorably or unfavorably, by the occupancy of other binding sites by the same or different substrate⁴⁵. The influence is mediated by an intramolecular network of short- and long-range interactions associated with conformational switching and energy transitions. This mechanism is observed in allosteric molecules, that is molecules which are characterized by having at least two ligand-binding sites: an orthosteric site and an allosteric site. Allosteric agents bind to pockets on target molecules, such as proteins, that do not overlap with the canonical, orthosteric binding pockets typically targeted by endogenous ligands⁴⁶. The binding of an allosteric agent influences the structural and/or functional state of the target. In this context, cooperativity is a special case of allostery associated with ligand-induced conformational dynamics, which may provide free energy of allosteric coupling via entropic effects⁴⁷.

Two or more agents/substrates involved in cooperative binding can be categorized as follows: i) they can be chemically distinct molecules, a concept denoted as “heterotropic cooperativity”, or ii) they can be two or more copies of the same (chemically identical) molecule, a phenomenon known as “homotropic cooperativity”⁴⁸⁴⁹. These categories provide the foundation for understanding the two principal types of allostery.

In heterotropic cooperativity, the interacting agents are chemically distinct from one another. One molecule serves as the effector or modulator, while the other acts as the primary ligand or substrate. The binding of the allosteric effector to a specific regulatory site (allosteric site) induces a conformational or dynamic change in the protein that alters the affinity or efficacy for the primary ligand at its functional site (typically the orthosteric site). This mechanism underlies heterotropic cooperativity, wherein the binding of one molecule influences the binding or activity of another, distinct molecule.

In homotropic allostery / homotropic cooperativity, the interacting agents are chemically identical -that is, multiple copies of the same ligand or substrate bind to multiple sites on the same protein or protein complex. The binding of the first molecule induces a conformational change that increases (positive cooperativity) or decreases (negative cooperativity) the affinity of the subsequent binding sites for additional molecules of the same type. This phenomenon is referred to as homotropic cooperativity, and is often observed in multimeric proteins with symmetrical or quasi-symmetrical binding sites. A classical example is hemoglobin, where the binding of oxygen to one heme site increases the affinity of the remaining sites for additional oxygen molecules — a hallmark of positive homotropic cooperativity.

Hence, in homotropic cooperativity, when one agent binds to a molecule, it influences the affinity of an additional copy or copies of the same agent/protomer for the same molecule. This type of allostery typically alters both the position and the shape of the binding curve. Positive homotropic cooperativity typically results in a more pronounced (steeper) response and higher-order dependence on ligand concentration, while negative homotropic cooperativity leads to a less pronounced (shallower) response and lower-order dependence on ligand concentration⁴⁸. Homotropic cooperativity is the primary mechanism by which evolution steepens the binding curves of biomolecular receptors to produce more responsive input-output behavior⁴⁸ . Consequently, a significant portion of biological processes are regulated by oligomeric ligandbinding proteins, such as hemoglobin and the p97 ATPase⁵⁰. Positive or negative homotropic cooperativity typically occurs when a protein binds its ligand with a stoichiometry higher than 1 :1. This can be the case when the protein is an oligomer composed of identical or similar subunits, allowing for multiple ligand binding sites. In such symmetric assemblies, even though the binding sites are identical, the ligand-binding affinities vary depending on the liganded state of the oligomeric protein. This means that a ligand's affinity for the receptor without bound ligands differs from its affinity for the same receptorwhen it is already interacting with one or more ligands⁵¹. Of note, the affinity of the oligomer for its primary ligand may additionally be regulated by effectors binding to sites different from that of the principal ligand (therefore introducing heterotropic regulation to the system).

In heterotropic cooperativity, the binding of one agent to a molecule influences the affinity of that molecule for a different agent at another site or epitope. This influence can increase (positive heterotropic cooperativity) or decrease (negative heterotropic cooperativity) said binding affinity. Consequently, the binding curve shifts toward lower or higher ligand concentrations, while the shape of the curve remains unchanged.

Cooperativity is often attributed to conformational changes in molecular structures. However, it has been shown that cooperative processes do not necessarily involve large conformational changes but can be transmitted through subtle alterations in protein motions⁵². Proteins are dynamic ensembles of conformations, in which allosteric motions occur even without ligand/modulator binding. However, ligand/modulator binding shifts the dynamic equilibrium by preferentially stabilizing a particular motion. Changes in free energy of a few kcal/mol can be easily achieved by a slight stiffening of a few of the many global dynamic modes available to a protein. Therefore, it is preferable to describe cooperativity both in terms of conformational changes (if observable) and thermodynamics, as cooperativity is fundamentally thermodynamic in nature⁵³.

Both global and cooperative (site-specific) binding events can be thermodynamically analyzed using isothermal titration calorimetry (ITC)⁵⁴, allowing for the characterization of the “entropyenthalpy compensation” phenomenon in protein-protein interactions. Such interactions are driven by favorable changes in free energy, which can involve an increase in entropy and/or a decrease in enthalpy⁵⁵. In this context, the enthalpy change reflects the strengthening of interactions between the ligand and receptor. These interactions encompass functional group interactions (ionic bonds, hydrogen bonds, van der Waals forces), conformational adjustments, polarization of interacting groups, and electrostatic complementarity. Entropy, on the other hand, can be described as a measure of disorder within a molecular system. Changes in binding entropy reflect alterations in the internal rotations and vibrations of molecules, exemplified by the loss or gain of motion. For instance, an increase in enthalpy due to tighter binding can lead to a reduction in entropy by limiting the mobility of the interacting molecules. Additionally, in protein-protein interactions, desolvation and counterion release upon complex formation can significantly impact the entropy value. The entropic contribution to free energy in receptor-ligand binding can be calculated using the interaction entropy method, as described in Duan etal., 2017^x, among other methods.

Factor a

In the context of this invention, the term “factor a,” “factor alpha,”, “a value”, "a factor" or simply “a” denotes a parameter used to quantify the interaction(s) between binding sites of a multisubunit protein complex, such as a ternary or a higher-order protein complex. Factor a indicates how the binding of one ligand/agent influences the binding affinity of an additional ligand/agent (or a plurality of these) to a protein complex, and is therefore a measure of “cooperative binding,” as defined herein. For a ternary complex, such as the complex of PD-1 »PD-L1 with an allosteric modulator described in this invention, the cooperativity factor a can be calculated as the ratio of the equilibrium dissociation constant (K_d) of the binary complex to the K_d of the ternary complex; as described in this application, K_d is a measure of the binding affinity between at least two molecules. Using this framework, the effect of the allosteric modulator of the PD-1 »PD-L1 complex, as a nonlimiting example disclosed herein, can be defined as the ratio of the K_d of the PD-1 »PD-L1 complex to the K_d of the ternary complex of PD-1 »PD-L1 ’allosteric modulator. In this example, the ratio (i.e. factor a) quantifies how the presence of the allosteric modulator affects the binding affinity between PD-1 and PD-L1.

For higher-order protein complexes involving more than 3 subunits, factor a can be generalized to account for the combined interactions among multiple binding sites. In such cases, a may be calculated by comparing the association constants of the complex formation in the presence and absence of other ligands. Here, the association constant (K_a) is understood as the reciprocal of the equilibrium dissociation constant (K_d), i.e., K_a = 1/K_d.

The term “positive cooperativity” (a >1) refers to a phenomenon observed in protein complexes where the binding of one molecule (e.g., a ligand or an agent) to a protein increases the affinity of subsequent molecules (ligands or agents) to other binding sites on the same protein. Thus, the binding of the first molecule enhances the subsequent binding of another molecule or molecules, resulting in a nonlinear relationship between the molecule (ligand/agent) concentration and binding activity. In this scenario, the binding sites interact such that the overall stability of the complex increases as more ligands bind. Such positive cooperative behaviour often stems from conformational changes induced by molecule binding, which facilitate the binding of additional molecule to the protein via reduction of the free energy of binding, as described in this application.

Conversely, “negative cooperativity” (a<1) indicates that the binding of one or more ligands or agents to a protein reduces the binding affinity of additional ligands or agents to that protein. In this case, the binding sites interact in a manner that decreases the overall stability of the complex as more ligands bind.

Notably, formation of a ternary complex often exhibits cooperativity in that the free energy change attendant to its formation is more negative or more positive than the sum ofthe free energy changes associated with the formation of the binary complexes. In this framework, positive cooperativity (a>1) increases and negative cooperativity (a<1 ) decreases the thermodynamic stability of the ternary complex⁵⁷.

Protein

Proteins are essential building blocks of living organisms, made up of polypeptide chains composed of amino acids, which translate the genetic information stored in DNA. Specifically, the term "protein" denotes one or more polypeptides functioning as a discrete unit. When a single polypeptide operates autonomously without requiring permanent or transient physical interaction with other polypeptides to form a functional entity, the terms "polypeptide" and "protein" are used interchangeably. Conversely, if the discrete functional unit comprises multiple polypeptides that physically interact, the term "protein" encompasses the assembly of polypeptides that are physically associated and operate collectively as a functional unit. In this context, the term "polypeptide" refers to any chain or chains of two or more amino acids, irrespective of the molecule’s length, wherein the amino acid residues are linked by covalent peptide bonds (i.e. amide bonds linking the amine of one amino acid to the carboxyl of another amino acid). Hence, "peptide", "polypeptide", "amino acid chain", or any other term used to describe a chain or chains of two or more amino acids, fall under the definition of a "protein". A "protein" or "polypeptide" may also refer to a partial amino acid sequence derived from its original molecule, for instance after enzymatic digestion (such as tryptic digestion).

Polypeptide-forming amino acids may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, amino acid isomers, unnatural amino acids, synthetic amino acid analogues, and variants thereof. Furthermore, the term "protein" is also intended to encompass products resulting from post-translational modifications of a polypeptide. Examples of such modifications include, but are not limited to, acetylation, acylation, adenylation, alkylation, amidation, arginylation, beta-lysine addition, biotinylation, butyrylation, carbamylation, carbonylation, citrullination, C-linked glycosylation, crotonylation, deamidation, diphthamide formation, eliminylation, ethanolamine phosphoglycerol attachment, farnesylation, flavinylation, formylation, gamma-carboxylation, geranylgeranylation, glutarylation, glutathionylation, glypiation, hydroxylation, hypusine formation, iodination, ISGylation, isoaspartate formation, isopeptide bond formation, isoprenylation, lipoylation, malonylation, methylation, myristylation, neddylation, nitration, N-linked glycosylation, nucleotide addition, O-GlcNAcylation, O-linked glycosylation, oxidation, palmitoylation, PARylation, PEGylation, phosphate ester (O-linked) or phosphoramidate (N-linked) formation, phosphopantetheinylation, phosphorylation, polyglutamylation, polyglycylation, prenylation, propionylation, protein splicing, proteolytic cleavage, pupylation, pyroglutamate formation, racemization, retinylidene Schiff base formation, S-nitrosylation, stearoylation, sulfation, sumoylation and ubiquitination. Finally, a polypeptide can originate from a natural biological source or be produced using recombinant methods, without necessarily being translated from a predetermined nucleic acid sequence. It can be produced using various methods, including chemical synthesis techniques such as solid-phase peptide synthesis (SPPS), thioester-forming ligation, oxime and hydrazone-forming ligation, thiazolidine/oxazolidine-forming ligation, and ligation by disulfide exchange (thioacid-capture ligation), among others.

Amino acid

The term "amino acid" encompasses all natural a-amino acids of the L or D series, each having the following "side chain": H for glycine, CH3 for alanine, CH(CH3)2 for valine, CH2CH(CH3)2 for leucine, CH(CH3)CH2CH3 for isoleucine, CH2OH for serine, CH(OH)CH3 for threonine, CH2SH for cysteine, CH2CH2SCH3 for methionine, CH2-(phenyl) for phenylalanine, CH2-(phenyl)-OH for tyrosine, CH2-(indole) for tryptophan, CH2COOH for aspartic acid, CH2C(O)(NH2) for asparagine, CH2CH2COOH for glutamic acid, CH2CH2C(O)NH2 for glutamine, CH2CH2CH2-N(H)C(NH2)NH for arginine, CH2-(imidazole) for histidine, CH2(CH2)3NH2 for lysine, and NH(CH2)3CHCOOH for proline. This includes the same side chains of amino acids with suitable protecting groups. Additionally, the term "amino acid" encompasses non-natural amino acids such as ornithine (Orn), norleucine (Nle), norvaline (NVa), 0-alanine, L or D a-phenylglycine (Phg), diaminopropionic acid, diaminobutyric acid, aminohydroxybutyric acid, and other synthetic amino acids known in the field of peptide chemistry. Table 2 lists amino acid names/codes and their abbreviations used in this application. Receptor

As used herein, the term “receptor” holds its conventional meaning in the field and denotes a protein located on the cell surface or within a cell, capable of receiving and transducing a chemical signal through ligand binding, thereby initiating a series of biochemical reactions that ultimately lead to a biological effect on the receptor-bearing cell. A receptor contains at least two active sites: i) one for recognizing and binding to a ligand, preferably a natural/endogenous ligand, such as an orthosteric agonistic ligand, and ii) another functionally active site that generates a biochemical response.

Receptors are classified into two categories based on their cellular localization: cell surface receptors and intracellular receptors. Cell-surface receptors also referred to herein as ‘surface receptors’ are receptor proteins exposed to the extracellular surface of a cell, and at least partially embedded in the cell membrane as to provide for a signal or connectivity to the intracellular components of the cell. There are currently three primary categories of cell surface receptors,: i) G protein-coupled receptors, ii) enzyme-coupled receptors, and iii) ion-channel-linked receptors. Each receptor class facilitates the transmission of extracellular signals through distinct mechanisms, many of which involve the initiation of specific protein-protein interaction cascades within a receptor-bearing cell. G protein-coupled receptors exert their intracellular effects by activating guanine nucleotide-binding proteins and specific effector enzymes. Enzyme-linked receptors can function either directly as enzymes or can activate associated enzymes upon their stimulation. This activation often leads to receptor autophosphorylation, resulting in the recruitment of associated proteins that act as intracellular signal transducers, lon-channel-linked receptors are proteins that convert chemical signals into a charge flux across the cell membrane by opening an intrinsic pore, allowing ions to flowthrough it⁵⁸.

According to various embodiments of the present invention, the term “receptor” specifically refers to an immune receptor that is a cell surface and/or membrane-bound receptor, preferably of the immunoinhibitory receptor type. The term "immunoinhibitory surface receptor" refers to a membrane-bound protein expressed on the surface of an immune cell that negatively regulates, attenuates, or suppresses immune cell activation, signaling, or effector function upon engagement with its cognate ligand. Such receptors typically contain one or more immunoreceptor tyrosinebased inhibitory motifs (ITIMs), immunoreceptor tyrosine-based switch motifs (ITSMs), or other inhibitory signaling domains within their cytoplasmic tails, and mediate immunosuppressive signaling through the recruitment of phosphatases or other negative regulators of immune signaling cascades. Exemplary immunoinhibitory surface receptors include, but are not limited to, PD-1 (programmed cell death protein 1 ), CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), LAG-3 (lymphocyte-activation gene 3), TIM-3 (T-cell immunoglobulin and mucin-domain containing-3), and TIGIT (T cell immunoreceptor with Ig and ITIM domains).

Immune receptor

An “immune receptor” denotes a specialized protein structure situated on the surface orwithin the cell membrane of an immune cell, capable of receiving and transmitting chemical signals via orthosteric ligand binding. Such binding instigates a response within the immune system. In general, the type and strength of an immune response is governed by the net balance of activating and inhibitory signals. This balance is influenced by the expression levels of activating and inhibitory receptors on the cell surface, as well as the availability of their corresponding ligands in the surrounding environment.

With regard to this matter, the term “immune cell” refers to a cell of hematopoietic origin that plays a role in an immune response and/or immune homeostasis, as known in the art. Hence, immune cells encompass, without limitation, all mature and immature forms of: i) lymphoid cells, including T cells (comprising helper, cytotoxic, and regulatory T cells), B cells, natural killer (NK) cells, innate lymphoid cells (ILCs), gamma delta T cells (y6 T cells), natural killer T (NKT) cells, and innate-like T cells (such as mucosal-associated invariantT (MAIT) cells and invariant natural killerT (iNKT) cells); and ii) myeloid cells, including macrophages, dendritic cells, neutrophils, monocytes, mast cells, eosinophils, and basophils. Importantly, the term “immune cell” refers to both naturally occurring and engineered/non-natu rally occurring immune cells, the latter group includingT cells comprising chimeric antigen receptors (CAR-T cells)⁵⁹, T cells expressing an engineered T cell receptor (TCR-T cells)⁶⁰, TCR-modified NK (TCR-NK) cells⁶¹, cytokine-induced killer (CIK) cells⁶², chimeric antigen receptor-modified macrophages (CAR-M cells)⁶³, and CXCL9/10-engineered dendritic cells⁶⁴, among others.

Ligand

In the context of this invention, the term “ligand”, “ligand of a receptor” or “receptor ligand”, as used interchangeably herein, refers to a molecule, preferably a peptide, protein, or a derivative, fragment or complex thereof, that exhibits specific binding affinity towards an orthosteric site of a target protein, i.e., a receptor protein. Such binding typically induces a conformational change in the target receptor and modifies its biological activity. The term "receptor agonist," also referred to herein as an "agonist," refers to a ligand that binds to a receptor and induces its functional activity, thereby initiating or promoting a physiological response associated with receptor activation. Conversely, a ligand that binds to a receptor without activating the physiological response is termed a “receptor antagonist”. In the current application, the term “ligand” encompasses naturally occurring/endogenous, recombinant, and synthetic ligands. In various embodiments of this invention, the ligand is preferably a “natural ligand” understood as an endogenously produced molecule that specifically binds to an immune receptor, as defined herein, to form a complex, as defined herein, and elicit a biological response, such as activation of downstream signaling.

In receptor-ligand interactions, a receptor and its orthosteric ligand constitute a complex-forming pair, associated by robust non-covalent bonds formed through interactions such as Van der Waals forces, hydrophobic interactions, n-n interactions, ionic interactions, or electrostatic forces. The nature of these interactions is contingent upon the structural and energetic compatibility of the receptor and ligand. The formation of receptor-ligand pairs is a multifaceted process and may involve various stages. When both the receptor and ligand are proteins, which is the most common instance of receptor-ligand complex formation, the process encompasses the following steps: 1 ) initial recognition of the receptor by the corresponding ligand from a distance, predominantly mediated by electrostatic forces; 2) reorientation and adjustment of the structural conformations to achieve optimal interface contact; and 3) physical binding of the two molecules⁶⁵. In this scenario, the complementarity between the surfaces of the binding proteins relies on both the shape/configu ration⁶⁶ and electrostatic properties of the binding sites⁶⁷⁶⁸. The kinetics of receptorligand binding is characterized by parameters encompassing the “binding affinity” and “kinetic rates”, as described here.

In general, receptor-ligand binding is driven by the differential free energy between the bound complex and the unbound molecules^{69 70}. The binding free energy comprises several energetic components, encompassing non-bonded factors such as electrostatic and van der Waals interactions, alongside bonded components like mechanical energy and entropy fluctuations⁷¹. Notably, these energy contributions are intricately linked to the protonation states and cannot be calculated independently of such states⁶⁵.

Ligand of an immune receptor

The term “ligand of an immune receptor” or “immune receptor ligand” encompasses molecules, preferably peptides, proteins, or their derivatives, fragments or complexes, which exhibit specific bindingto receptor proteins localized on the surface or within the cell membrane of immune cells. These ligands may originate from various cellular sources, including i) target receptor-bearing immune cells themselves, ii) other immune cells, or iii) non-immune cells such as cancer cells. Notably, a ligand of an immune receptor may exist as a membrane-bound or soluble molecule. As non-limiting example provided herein, in the case of the PD-1 receptor expressed on T cells, its ligands PD-L1 and PD-L2 are typically membrane-bou nd proteins found on antigen-presenting cells (APCs) or other immune cells. When PD-1 interacts with these ligands, it attenuates T cell activation, thereby regulating immune responses and maintaining peripheral tolerance. Similarly, the CTLA-4 receptor interacts with ligands CD80 and CD86, which are commonly present on the surface of APCs. Binding of CTLA-4 to these ligands also suppresses T cell activation, thereby contributing to immune homeostasis and preventing “autoreactivity”, as further defined in this application.

To bind

The terms “bind”, “bound”, “binding,” and their derivatives refer to covalent or non-covalent interactions between two or more molecules, such as proteins or their fragments, resulting in the association of the interacting molecules. In this context, a “binding site” or “binding pocket” is a region of a protein that interacts with another molecule (e.g., protein, peptide, antibody, Nanobody, etc.) or with a part of another molecule. A binding site includes residues or atoms that a ligand couples with through various interactions, such as ionic, electrostatic, hydrophobic, hydrogen bonding, or Van der Waals interactions, among others. Typically, this region comprises amino acid residues directly involved in binding. It may also include residues that, while not directly involved in binding, are interspersed between interacting residues and/or provide structural, spatial, energetic, and/or other functions.

Binding affinity

The term “affinity” or “binding affinity” refers to the strength of a reversible interaction between at least two molecules, such as a ligand and a receptor. In general, a high-affinity interaction elicits a robust biological response, whereas a weak-affinity interaction induces a minimal biological response⁷². For example, a simple reversible reaction between ligand L and receptor R can be

K_on described by the following equation: [L] [R] [LR]. In this scenario, [L] and [R] represent the

^KOff concentrations of the ligand and receptor, respectively, [LR] denotes the concentration of their bound complex L»R, K_on represents the association rate constant measured in M'¹s^-1, and K_off represents the dissociation rate constant measured in M'¹s^-1; here, “M'¹s^-1” stands for “per molarity per second”. K_on and K_off are kinetic rates which respectively measure the rate of bond formation and the rate of bond dissociation. When the system is at equilibrium, the ratio of the rate constants can be solved, and the binding affinity can be measured as “equilibrium dissociation constant” (K_d):

K_d = Additionally, the receptor-ligand bond lifetime T is the inverse value of the K_off <m rate, i.e., T = 1/K_off. For such binary complex systems, the saturation binding curves adopt a sigmoidal shape⁷³. Likewise, the equilibrium dissociation constant can also be defined for ternary complexes, such as in the case where the allosteric modulator of the present invention is bound to a binary receptorligand complex, such as the PD-1 »PD-L1 complex. For instance, a binding reaction between a receptor/?, its ligand L, and an allosteric modulatorM, leadingtotheformation of a ternary complex R»L»M, can be described by the following three independent equilibrium constants:

Herein, “a”, “factor a” or “factor alpha”, as used interchangeably, denotes the “cooperativity factor” defined as the ratio between the ternary equilibrium dissociation constant and the corresponding binary equilibrium dissociation constant⁷⁴. An a value greater than 1 indicates positive cooperativity, an avalue lowerthan 1 indicates negative cooperativity, whereas an avalue of 1 indicates no cooperativity between the first and second binding events. For such ternary complex systems at equilibrium, the concentration of the ternary complex [RLM] can be described as the function of the free ligand concentration [L]: _wp_ere ([L]) =

[ ?_t] + [M_t] + + K_R1) ([L] + K_M1). Here, [R_t] and [M_t] are total concentrations of the receptor and allosteric modulator, respectively.

Notably, these equations apply universally to all ternary complex systems regardless of the cooperativity status⁷³.

At a fundamental level, a protein-ligand interaction takes place in a thermodynamic system composed of the solute (the protein and ligand molecules) and the solvent (such as the extracellularfluid). In such a system, interactions and heat exchange amongthe agents, molecules or substances involved are governed by the laws of thermodynamics⁷⁵. The Gibbs free energy (G), a key thermodynamic potential that measures the maximum reversible work a system can perform under constant temperature and pressure conditions (i . e. , isothermal and isobaric conditions), is crucial for understanding these driving forces⁷⁶. Herein, the change in Gibbs free energy (AG) determines whether the interaction between a receptor and ligand can occur spontaneously. AG takes into account both the enthalpic and entropic contributions to the system and is calculated using the formula: AG = AH - TAS, where AH and AS denote the change in enthalpy and entropy of the system upon ligand binding, respectively, and T is the temperature in Kelvin. Alternatively, at any moment in time during receptor-ligand association, AG can be given as: AG = AG° + RT InQ. Here, AG° represents the “standard binding free energy”, indicating the free energy change measured under the conditions of 1atm pressure, temperature of 298K, and the effective reactant (protein and ligand) concentrations of 1 M; R is the universal gas constant (1 .987 cal/K- mol); Tis the temperature in degrees of Kelvin; and Q is the reaction quotient. The reaction quotient is defined as the ratio of the concentration of the protein-ligand complex to the product of the concentrations of the free protein and free ligand at any given moment in time.

Analogous to any spontaneous process, protein-ligand binding occurs only when AG of the system is negative at equilibrium under constant pressure and temperature. Thus, a negative AG indicates a spontaneous binding process (favorable interaction), where the formation of the receptor-ligand complex releases free energy and leads to increased complex stability. Conversely, a positive AG indicates a non-spontaneous process (unfavorable interaction), whereby external energy input is required to form or maintain a complex. Consequently, the extent of protein-ligand association can be determined by the magnitude of AG, which indicates the stability of the protein-ligand complex, or alternatively, the “binding affinity” of a ligand to a specific acceptor (such as a receptor). Notably, the free energy specifically depends on the system's states, indicating that AG is governed solely by the initial and final thermodynamic states, regardless of the pathwaythat connects these states. Therefore, when Q = K_b = -^^22L, the association reaction is at equilibrium, and AG = 0.

^Koff

Additionally, the “affinity” or “binding affinity” can also be explained in terms of the Gibbs free energy of dissociation (AG_d), which, for an interaction to occur, must be positive: AG_d = — RT In— = AH_d — TAS_d. Here, C_o is the concentration that defines the standard state, set at 1 co mol/l by conventional criteria, R is the gas constant, with a value of 8.3144 J 1/K- mol, T is the absolute temperature in Kelvin, and AH_d, AS_d, and AG_d represent the changes in enthalpy, entropy, and binding free energy upon complex dissociation, respectively. The binding affinity is related to the Gibbs free energy of association (AG_a) in the following way: AG_a = — G_d. The free energy of binding, AG_a, and the free energy of dissociation, AG_d, describe all the chemical and energetic factors involved in the dissociation and association reactions, respectively. AG_a consists of two opposing general energies: one that favors the complexation of the unbound partners and one that favors the unbound state: AG_a = AG_bond + AG_entropy, where AG_bond denotes the intrinsic nonbonded interaction energy that includes all chemical forces acting on the interface of the complex, and AG_entropy denotes entropy change, analogous to the physical enthalpy and entropy changes.

In light of the above, protein- protein interactions are driven byfavorable changes in the free energy, which may include an increase in system entropy and/or a decrease in system enthalpy⁵⁵. Enthalpy represents the total energy of a thermodynamic system, encompassing the internal energies of both solute and solvent, as well as the energy required to create space for the system (calculated as the product of the system volume and pressure)⁷⁷. The change in enthalpy, AH, can be negative in exothermic processes, such as the formation of energetically favorable noncovalent interactions between atoms, and positive in endothermic processes, which involve the disruption of such interactions. In the context of this disclosure, AH specifically reflects the energy change when a ligand binds to a receptor. Such energy changes result from the formation of noncovalent interactions (including van der Waals contacts, hydrogen bonds, and ion interactions) at the binding interface. However, the heat effect of a binding reaction is a global property, encompassing contributions from both the solute and the solvent⁷⁸. Moreover, the formation of favorable interactions often necessitates the disruption of pre-existing ones. The change in enthalpy upon binding is influenced by multiple interactions: the loss of hydrogen bonds and van der Waals interactions between the protein and solvent, the formation of noncovalent interactions between the protein and ligand, and the reorganization of solvent molecules near the complex surfaces. Each of these components may contribute either positively or negatively, and the overall change in enthalpy is determined by the combined effect of these contributions⁷⁹.

Entropy, on the other hand, quantifies the distribution of heat energy throughout a thermodynamic system. According to the second law of thermodynamics, heat spontaneously flows from regions of higher temperature to regions of lower temperature, reducing the system's initial order⁸⁰. Therefore, entropy can be understood as a measure of the disorder or randomness of atoms and molecules within a system. AS is a global thermodynamic property indicating overall increases or decreases in the system's freedom, denoted by positive and negative signs, respectively. The total entropy change associated with binding (AS) can be parsed into three entropic components: AS = AS_solv + AS_conf + AS_r/t. ASsdv accounts for solvent entropy changes due to surface burial upon binding, often beneficial due to its typically positive value; AS_conf reflects changes in the flexibility of the protein and ligand upon binding, influencing the binding entropy either positively or negatively, depending on how the degrees of freedom are altered in the complex⁸¹; and AS_r/_t represents the loss of translational and rotational degrees of freedom upon complex formation, generally unfavorably affecting the binding entropy. These components collectively influence the net entropy change, with a positive net change contributing favorably to the binding free energy and a negative net change contributing unfavorably. Successful binding reactions typically mitigate entropic penalties, such as negative AS_r/t^82,83, through substantial solvent entropy gains (AS_soiv) or favorable protein-ligand interactions (resulting in negative AH)⁷⁵. Overall, two thermodynamic quantities, enthalpy change (AH) and entropy change (AS), determine the sign and magnitude of the binding free energy (AG). Therefore, AH and AS are considered the driving factors for protein-ligand binding. The contributions of AH and AS to AG are interrelated. For example, tight binding due to multiple favourable noncovalent interactions between the interacting partners results in a large negative AH. However, this is often accompanied by a negative AS due to restricted mobility of the complexed protein, leading to a moderate change in AG⁸⁴. Similarly, a large entropy gain is typically paired with an enthalpic penalty (positive AH) due to the energy needed to disrupt noncovalent interactions. This phenomenon, where medium-magnitude free energy change arises from complementary changes between AH and AS, is known as the “enthalpyentropy compensation”⁸⁵.

The 3D structure of a protein-protein complex allows for the dissection of its various energetic components, such as bond lengths, bond angles, and van der Waals interactions. Estimating the binding energy involves calculating the difference in these components between the separated proteins and the complex⁸⁶. Considering this perspective, molecular dynamics (MD) simulations can be employed to distinguish between the bound and unbound energetic states of proteins. The ensemble of structures generated by MD simulations provides insights into protein dynamics and enables the calculation of the average interaction energy. Sampling this ensemble allows for the calculation of probability distributions of energetic states, which can be used to estimate configurational entropy. An increase in the number of structural and energetic states upon complex formation corresponds to an increase in configurational entropy, contributing favorably to the free energy of binding⁵⁵.

Affinity between two or more molecules, such as an antibody (or nanobody) and an antigen, or components within a signaling complex, such as an immune receptor and a ligand, can be assessed directly, as described above, or indirectly. Indirect methods often rely on surrogate properties that indicate or correlate with the binding affinity. These surrogate indicators include the quantity or level of binding between the components, and biophysical characteristics predictive of binding strength, such as molecular charge, rotational activity, diffusion rate, melting temperature, electrostatic interactions, and conformational changes. Additionally, stability under varying conditions of temperature, pH, or ionic strength can serve as informative measures. These indirect approaches can provide valuable insights into molecular interactions without requiring direct affinity measurements, such as those performed using techniques like isothermal titration calorimetry or surface plasmon resonance. Furthermore, the term “apparent affinity” refers to the measurement of the strength of binding interactions between two or more molecular entities under conditions where the binding affinity is affected by factors such as allosteric modulators, inhibitors, binding component valency, and other factors present in the binding reaction.

To specifically bind

Molecular recognition refers to the process by which biological molecules, such as proteins, bind with each other through covalent or non-covalent interactions to form specific complexes. This process is characterized by two key features: (i) specificity, which relates to the degree of preference towards a particular target; and (ii) affinity, which is the measure of the binding strength between two interacting partners. A specific binding partner with high affinity remains bound even in the presence of high concentrations of less specific partners characterized by lower affinity for the target⁷⁵.

In this context, the term “specifically binds” and its derivatives describe the ability of a binding agent, as defined in this application, to bind to a specific target with high degree of recognition and association, while displaying lower, limited or no recognition and/or binding to other targets. Yet, specific binding does not necessarily imply exclusive binding, as the binding agent may still interact with other molecules to some extent. However, an agent that specifically binds to a defined target or binding site or epitope of said target demonstrates a significantly stronger preference for that target or binding site or epitope of said target compared to other targets. Within the context of the invention, the term “specifically binds” particularly pertains to the capacity of an allosteric modulator to selectively recognize and/or bind to a protein complex as a whole entity, rather than to its individual constituents/subunits. For example, the allosteric modulator of the PD-1 »PD-L1 complex, as disclosed here, exhibits specific binding to an allosteric epitope of said complex. This allosteric epitope arises from conformational changes induced by the orthosteric interaction between PD-1 and PD-L1 . A "conformational epitope" comprises residues that are non-contiguous in the protein sequence but spatially proximal in the three-dimensional (3D) structure of the protein or protein complex, forming an antigenic surface⁸⁷. Such antigenic surfaces of immune receptorligand complexes enable specific binding of the allosteric modulators disclosed in this application.

Conformational epitope

The term "conformational epitope" when used in reference to a protein complex, refers to a three- dimensional antigenic determinant formed by amino acid residues contributed from two or more distinct protein subunits or chains within the protein complex. These residues are spatially juxtaposed upon quaternary structure formation, resulting in an epitope that is absent or only partially present in the individual subunits when isolated. Such conformational epitopes depend on the native assembly and interface architecture of the protein complex, and may be recognized by antibodies or immune receptors or allosteric modulators specifically targeting the assembled complex. Recognition or specific binding of these epitopes requires the intact, properly folded multi-subunit complex, and disruption or dissociation of the complex typically abolishes epitope integrity and binding, or at least significantly reduces said binding.

Protein complex

The term “protein complex”, as used herein, designates a cluster of molecules comprising at least one protein, stabilized by covalent and/or non-covalent bonds (such as Van der Waals forces, hydrophobic, and hydrophilic interactions). A protein complex can be composed entirely of proteins or include other types of molecules, such as carbohydrates, lipids, glycolipids, nucleic acids, oligonucleotides, nucleoproteins, nucleosides, nucleoside phosphates, and other entities. Hereby, a “protein complex” may specifically refer to an association of two or more proteins interacting non-covalently, without involvement of other types of molecules, termed a “proteinprotein complex”. In preferred embodiments, a protein complex is formed by a receptor and a ligand, or a plurality of these. Furthermore, within the scope of this invention, a “protein complex” preferably denotes an assembly of molecules that can form in vivo under physiological and/or pathophysiological conditions. Each molecule engaged in a protein complex is referred to as a “constituent”, “individual constituent”, “element”, “individual element”, “subunit”, “individual subunit”, “member”, “individual member” or “protomer”. A protein complex is homomeric when all its subunits consist of the same type of protein, whereas heteromeric complexes consist of different constituents. Moreover, assemblies of protein complexes can result in homo-multimeric or hetero-multimeric complexes, characterized by interactions that range from transient to stable. Generally, multiprotein complexes form by stochastic interactions between mature proteins, via chaperone-facilitated interactions, or through co-translational mechanisms⁸⁸.

Conformation

The term “conformation” or “conformational state”, as used in this application, relates to the range of three-dimensional (3D) structures/shapes that a protein or protein complex may adopt. Protein conformation is defined as the spatial arrangement of atoms within its molecule, determining its overall geometric shape. A protein can be characterized by its hierarchical levels of assembly, which include primary, secondary, and tertiary structures; additionally, some proteins exhibit a quaternary structure. The primary structure consists of a linear sequence of amino acids, which are linked to each other through covalent peptide bonds. The secondary structure comprises regions of the amino acid chain stabilized by hydrogen bonds within the polypeptide backbone, resulting in alpha-helices and beta-pleated sheets. The tertiary structure refers to the overall 3D shape of a protein, determined by interactions among the side chains of the amino acids. The quaternary structure, which also impacts the 3D shape of a protein, arises from interactions between the side chains of two or more polypeptide subunits. Hence, the folding of a protein chain is constrained by covalent bonds between amino acids and various types of weak noncovalent bonds that form between different parts of the chain. These involve atoms in both the polypeptide backbone and the amino acid side chains. The weak noncovalent bonds include hydrogen bonds, ionic bonds, and van der Waals interactions. Individually, noncovalent bonds are 30-300 times weaker than the typical covalent bonds that form organic molecules. However, the cumulative effect of many weak bonds can significantly stabilize the folded structure by holding two regions of a polypeptide chain tightly together. A fourth weak force, hydrophobic interactions, also plays a central role in determining the protein shape. Hydrophobic molecules, including the nonpolar side chains of certain amino acids, tend to aggregate in an aqueous environment to minimize their disruptive effect on the hydrogen-bonded network of water molecules. Consequently, an important factor in protein folding is the distribution of its polar and nonpolar amino acids. Nonpolar (hydrophobic) side chains, such as those of phenylalanine, leucine, valine, and tryptophan, typically cluster in the interior of the molecule, away from the surrounding water in the cellular environment. In contrast, polar side chains, such as those of arginine, glutamine, and histidine, tend to arrange themselves near the surface of the molecule, where they can form hydrogen bonds with water and other polar molecules. When polar amino acids are buried within a protein, they are usually hydrogen-bonded to other polar amino acids or to the polypeptide backbone. As a result of these interactions, each protein assumes a specific 3D structure, or conformation. The final shape adopted by any polypeptide chain is typically the one that minimizes its free energy.

Notably, posttranslational and other modifications of a polypeptide chain, such as ligand binding (including the binding of allosteric modulators), phosphorylation, sulfation, glycosylation, or attachments of hydrophobic groups, among others, can influence the 3D structure of a protein. In this context, the bindingof a ligand induces reshapingof the protein's conformation. The interacting partners induce reciprocal conformational changes during the binding process that are necessary for specific recognition and binding, as described earlier. In principle, all molecular recognition processes require some degree of conformational adaptation⁸⁹. The structural changes due to ligand binding can vary from large, collective movements, such as loop closure over the binding pocket, to small, local fluctuations of side chains⁹⁰. Furthermore, environmental factors, such as acidity (pH), salt concentration, ionic strength, and osmolality of the surrounding solution, as well as interactions with other molecules, can affect protein conformation. The conformational state of a protein can be determined through functional assays measuring its activity or binding to another molecule, as well as through physical methods such as X-ray crystallography, nuclear magnetic resonance (NMR), cryo-electron microscopy (cryoEM), spin labeling, and other techniques recognized in the field.

Protein structures observed in various functional states frequently exhibit distinct conformations. Another layer of complexity stems from the fact that proteins in solution typically assume a spectrum of conformations, termed “ensemble”⁹¹. The conformational selection paradigm suggests that this ensemble encompasses a variety of conformations, some of which correspond to different functional states. Importantly, functionally relevant conformations may occupy only a small fraction of all possible conformations within an ensemble⁹². Furthermore, the binding of ligands or effectors can influence the ensemble, favoring the adoption of functionally significant conformations.

The degree of conformational sampling within a stable or metastable state is quantified by conformational entropy. This entropy is intricately linked to the shape of the free energy basin in the conformational space. In addition to thermodynamic properties, dynamic characteristics associated with time-dependent processes are also essential observables. These include timescales, magnitudes, and spatio-temporal correlations of internal motions, as well as the rates of conformational transitions. At the microscopic level, such dynamic properties are governed by the equations of motion and are influenced not only by free energy basins but also by barriers and other dynamic parameters, such as effective diffusion coefficients³⁹.

Conformation unique to a protein complex

The term “conformation unique to a protein complex” or “conformation specific to a protein complex” refers to a distinct 3D arrangement of atoms within a protein complex, which is formed when molecules engaged in the complex bind together. This unique complex conformation can differ significantly from the conformations of the individual constituent proteins when they are not part of the complex.

A unique 3D structure is critical for the biological function, stability, specificity, and/or affinity of a protein complex in binding to other molecules, such as ligands, substrates, or allosteric modulators. The term “conformation unique or specific to a protein complex” herein typically refers to the 3D structures of complexes formed by immune receptors and their ligands, which are distinct from the individual/unbound three-dimensionalstructures of these immune receptors and ligands. Thus, the term “unique" or "specific” refers to the fact that the conformation of these protein complex constituents/subunits does not exist when the constituents/subunits are unbound and exist as individual molecules or are part of a complex with different constituents/subunits. Complex-specific conformations are thus referred to herein as conformational states of the protein partners of said complex that are unique or exclusively present when the complex is formed, i.e. when the individual protomers of the complex interact under a certain state or condition. The allosteric modulators as described herein are typically specifically binding to a binding site or epitopethat is unique or exclusively present when the protein complex is formed, i.e. fora complexspecific conformational epitope. Though, it is referred to herein that the allosteric modulators ‘predominantly’ bind to said complex-specific conformation, wherein the term ‘predominantly’ is defined as that the binding site is present on the protein complex, though in some cases, the binding site may still be partially present on the individual protomers, which may allow for residual binding, typically to a much lower extent, for the individual protein partners.

To induce

The terms “induce”, “induction” or “inducing”, as used herein, refer to causing, triggering, enhancing, or bringing about a specific effect, action or result, through direct or indirect means.

Inhibitory immune receptor

In the context of this invention, the term “inhibitory immune receptor” or “immune-inhibitory receptor” or “immunoinhibitory receptor” refers to a protein receptor present on the surface or within the membrane of an immune cell that negatively regulates or inhibits an immune response, often via attenuating stimulatory signals initiated by other receptors. A substantial body of biological evidence supports the role of inhibitory receptors in maintaining immune homeostasis and tolerance, thereby preventing pathological states. For example, murine models deficient in specific inhibitory receptors exhibit a heightened susceptibility to autoimmune diseases and increased immunopathology during infection⁹³. In humans, genetic correlations observed between autoimmune diseases and individual inhibitory receptors, or the downstream effectors of their signaling pathways, underscore the pivotal role these receptors play in the prevention and/or suppression of autoreactivity¹¹’⁹³.

The human genome encodes more than 300 genes encoding potential immune-inhibitory receptors⁹⁴. Most of these receptors contain one or more immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic tails. These motifs serve as docking sites for downstream effectors that mediate the inhibition of cellular action, such as proliferation, differentiation, and cytotoxicity. ITIM sequences consist of the consensus amino acid sequence (V- L-l-S)-x-Y-x-x-(l-L-V) (SEQ ID NO: 1), where ‘x’ can be any amino acid. This domain can be extended to (V-L-l-S-T)-x-Y-x-x-(l-L-V) (SEQ ID NO: 2) to also include ITSM (immunoreceptor tyrosine-based switch motif) sequences, as there are inhibitory receptors, such as PD-1 , that rely partly on the ITSM for their inhibitory function⁹⁵. Upon ligand binding to the receptor bearing the ITIM, the tyrosine residue within the motif becomes phosphorylated, facilitating the recruitment of Src-homology 2 (SH2) domain-containing phosphatases such as SH2 domain-containing protein tyrosine phosphatase 1 (SHP-1 ), Src homology region 2 domain-containing phosphatase-2 (SHP-2), Skicontaining inositol phosphatase-1 (SHIP)⁹⁶, or the inhibitory C-terminal Src kinase (CSK)^97-99. Certain inhibitory receptors, such as the CD200 receptor (CD200R) and cytotoxic T-lymphocyte antigen-4 (CTLA-4), utilize alternative signaling motifs to exert their regulatory functions. These include N-P-x-Y (SEQ ID NO: 3), E-E-D-E-x-x-P-Y-x-x-Y-x-x-K-x-N-x-x-Y (SEQ ID NO: 4), wherein ‘x’ may be any amino acid residue, and KIEELE (SEQ ID NO: 5), among others^{100 101}.

Inhibitory immune receptors exert their effects locally either by disrupting stimulatory receptors, thereby hindering cell activation, or by responding to local stimulatory signals themselves. Local disruption of stimulatory receptors entails the recruitment of inhibitory molecules to TOR clusters; these inhibitory molecules include phosphatases such as SHP-1 and SHP-2. The recruitment initiates localized dephosphorylation resulting in immune inhibition. Additionally, inhibitory immune receptors can undergo localized “priming” by signals present at sites of stimulation. For example, intracellular inhibitory motifs within PD-1 are phosphorylated by Lek, which accumulates at stimulated TCR clusters, thereby triggering localized inhibitory signaling¹⁰².

Importantly, inhibitory receptors function not as a binary on/off switches but rather as a “volume control” systems¹⁰³. This means that the effect of inhibitory receptor signaling on cellular function depends on the strength of the inhibitory signal relative to the activation signal in a specific time frame¹⁰⁴. In systems with both inhibitory and stimulatory receptors, the strength of the signals sensed by an immune cell is determined by receptor expression, ligand expression, the affinity of receptor-ligand complexes, and the degree of receptor crosslinking. The expression of inhibitory receptors and their ligands varies between tissues, cell types, and cell subsets, and can change based on the cellular activation status. Consequently, inhibitory receptors can fine-tune immune responses with high specificity¹⁰³.

During an immune response, a single cell can experience multiple activation events. In this regard, inhibitory receptors fall into two major classes with distinct functions: i) “threshold' receptors”, which are expressed before a cell is activated, and ii) “negative feedback” receptors, whose expression is induced in response to stimulatory signals. An exemplary inhibitory receptor functioning in the threshold mode is signal-regulatory protein-a (SIRPa), also known as SHPS1. SIRPa is consistently expressed in both resting and activated myeloid cells. It interacts with CD47, a protein expressed on the surface of most cells. The SIRPa-CD47 interaction generates a “don't eat me” signal, which inhibits the phagocytosis of CD47-expressing cells by myeloid cells¹⁰⁵. Poliovirus receptor (PVR; also known as CD155), on the other hand, is a negative feedback receptor upregulated on dendritic cells following TLR stimulation or upon CD4OCD40L complex formation¹⁰⁶.

It should be understood that many inhibitory receptors exhibit characteristics of both aforementioned categories to some extent and therefore may act as “threshold receptors” in one cell type or during one stage of cell differentiation and as “negative feedback receptors” in other cell types or during other stages of differentiation. For example, PD-1 expression is induced upon activation of naive T cells, where it acts as a negative feedback receptor¹⁰⁷. Conversely, PD-1 is also highly expressed on effector T cells¹⁰⁸, where it functions as a threshold receptor. In line with this, PD-1 is involved in mediating ? cell exhaustion¹⁰⁹. Consequently, immune checkpoint inhibition targeting PD-1 can be viewed as a strategy to lower the activation threshold of effector T cells by blocking the highly expressed PD-1. Furthermore, the expression of PD-1 's ligand, PD-L1 , is induced by IFNy, and therefore PD-L1 can be considered a “negative feedback ligand”. Hence, PD- 1 »PD-L1 signaling has characteristics of both a threshold and a negative feedback regulatory axis.

Another example of a dual-mode inhibitory receptor is CTLA-4. It acts as a negative feedback receptor on conventional ? cells, which upregulate its expression upon activation¹¹⁰. Conversely, CTLA-4 is constitutively expressed on regulatory T cells, where it may operate as a threshold receptor. Furthermore, the inhibitory collagen receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1 ) is not present in resting granulocytes but is induced when cells become activated, thereby acting as a negative feedback receptor. On the other hand, LAIR-1 is expressed in resting ? cells, where it serves as a threshold receptor¹¹¹. Of note, some receptors can exhibit characteristics of both categories within the same cell type: they are expressed before activation and are further upregulated after activation, thus fulfilling both the roles of setting a threshold and providing a negative feedback¹⁰³.

Immune checkpoint inhibition

The term “immune checkpoint inhibition” or “immune checkpoint blockade” refers to a therapeutic strategy or intervention that targets one or more immune checkpoint proteins to modify the regulation of an immune response. The targeting involves counteractingthe function, disruptingthe structure and/or modifying the localization of at least one immune checkpoint protein. This approach can lead to down-modulation, inhibition, up-modulation, or activation of an immune response. Immune checkpoint inhibition can be achieved through the use of specific immune checkpoint inhibitors or blockers. These agents are well-known in the art and include, without limitation: Pembrolizumab (Keytruda®), which targets PD-1 ; Tremelimumab (Imjudo®), which targets CTLA-4; Durvalumab (Imfinzi®), which targets the PD-1 »PD-L1 interaction; Nivolumab (Opdivo®), which targets PD-1 ; and Relatlimab, which targets LAG-3. These immune checkpoint inhibitors exhibit an opposite effect on immune receptor signaling and/or ligand binding compared to the allosteric modulators described in this invention.

Protein-protein interactors

The terms “protein-protein interactor” and “protein-protein interaction (PPI),” and derived terms thereof, are used to describe the association between at least two proteins that bind through covalent and/or non-covalent bonds to form a protein complex. A “protein-protein interactor” specifically refers to a protein that participates in this binding process, engaging with another protein to establish a structural connection, which may also be a functional connection. These interactions can involve proteins that are identical (homotypic interactions) or different (heterotypic interactions). Non-covalent bonds facilitating protein-protein interactions include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions. Such interactions are crucial for various biological processes, including immune responses, transmembrane signal transduction, and maintenance of cellular structural integrity.

To modulate

The terms “modulate”, “modulating”, “modulator” or “modulation” referto inducing a change from the baseline or current state, particularly involving positive or negative regulation or adjustment of normal functioning or activity relative to a reference, control or normal state. This results in a change in function or activity compared to the control condition (i.e., in the absence of the modulator or compared to a non-modulating control).

In the context of this invention, these terms can refer to various actions such as increasing, decreasing, masking, altering, overriding, boosting, or restoring the baseline functioning or activity of a receptor or receptor-ligand complex. For instance, baseline functioning or activity refers to the default signaling of a receptor-ligand complex, such as for the non-limiting example of the PD- 1 »PD-L1 complex, in the absence of any external modulator affecting such signaling. The signaling activity may be characterized by its strength, duration, and other features that could be subject to modulation.

In various embodiments of this invention, "modulation" of a protein complex occurs through: 1 ) binding of an agentto said protein complex, and 2) induction of conformational changes within the complex, leading to 3) altered cooperativity within the complex, which results in 4) changes in affinity between constituents of the complex, thereby 5) prolonging and/or potentiating downstream signaling for some or multiple aspects of the protein complex signaling pathways.

Particular embodiments described herein refer to “allosteric modulation” of a protein complex, preferably a ligand-receptor protein complex, such as PD-1 »PD-L1 or PD-1 »PD-L2, wherein said allosteric modulation occurs via: 1 ) binding of an allosteric agent to said protein complex, and 2) induction of conformational changes within the complex, leading to 3) induction of positive cooperativity within the complex, resulting in 4) increased affinity of the constituents of said complex, ensuing 5) prolonged or potentiated immunosuppressive signaling.

Antibody

The term “antibody” refers to an immunoglobulin (Ig) molecule or a molecule containing an Ig domain, which specifically binds with an antigen, including multimers thereof. In humans, antibodies are naturally produced by plasma cells to mediate an adaptive immune response against invading pathogens. Antibodies exist as one or more copies of a Y-shaped unit composed of four polypeptide chains. Each Y unit contains two identical heavy chains (H) and two identical light chains (L), which differ in their sequence and length. The top of the Y shape contains the variable region (V), also known as the fragment antigen-binding region (F(ab)), which binds tightly to a specific part of an antigen called an epitope. The base of the antibody consists of constant domains (C) that form the fragment crystallizable (Fc) region. This Fc region is essential for the antibody's function during an immune response. The Y-shape of an antibody can be cleaved into three fragments by the proteolytic enzyme pepsin: two F(ab) regions and one Fc region. The F(ab) regions contain the variable domains that bind to specific antigens. The Fc fragment provides a binding site for endogenous Fc receptors on the surface of lymphocytes and secondary antibodies. The type of heavy chain defines the overall class or isotype of an antibody. Mammals have five types of immunoglobulin (Ig) heavy chains, denoted by the Greek letters a, 6, s, y, and p. These correspond to the IgA, IgD, IgE, IgG, and IgM antibodies, respectively. Heavy chains vary in size and composition: a and y chains have approximately 450 amino acids, while p and s chains contain about 550 amino acids. Each heavy chain comprises two regions: a constant region (CH) and a variable region (VH). The constant region is identical in all antibodies of the same isotype but differs among different isotypes. The y, a, and 5 heavy chains have a constant region composed of three tandem Ig domains - CH1 , CH2, and CH3 - and include a hinge region for added flexibility. In contrast, the p and s heavy chains have a constant region composed of four Ig domains. The variable region (VH) of the heavy chain differs among antibodies produced by different B cells but remains identical for all antibodies derived from a single B cell or B cell clone. The variable region is approximately 110 amino acids long and consists of a single Ig domain. Mammals have two types of light chains, lambda (A) and kappa (K), which differ slightly in their polypeptide sequences. Each light chain consists of two domains: a constant domain (CL) and a variable domain (VL). The length of a light chain ranges from approximately 211 to 217 amino acids. An antibody contains two identical light chains. Other types of light chains, such as the iota (i) chain, are found in lower vertebrates like Chondrichthyes and Teleostei. The F(ab) region of an antibody contains the antigen-binding site known as the paratope. The paratope binds to a specific part of an antigen called the epitope, which is a small segment of the antigen, sometimes just a few amino acids in length. The paratope and epitope are typically held together by complementary shapes and intermolecular interactions, including Van der Waals forces, hydrogen bonds, electrostatic interactions, and hydrophobic interactions. The strength of these forces determines the antibody's affinity for the antigen. Antibodies can be intact immunoglobulins or immunoreactive portions of intact immunoglobulins. The term encompasses antibodies produced naturally, recombinantly, semi-synthetically, or synthetically. For example, an antibody may occur naturally, being produced or expressed endogenously by a cell or tissue and optionally isolated therefrom. Alternatively, an antibody may be recombinant, produced using recombinant DNA technology, and/or synthesized chemically or biochemically, in whole or in part.

Active antibody fragment

The terms "active antibody fragment", "antibody fragment", "antigen-binding fragment", and "functional antibody fragment" refer to a portion of any antibody or antibody-like structure that by itself has high affinity for an antigenic determinant, or epitope, and contains one or more complementarity-determining-regions (CDRs) accounting for such specificity. Non-limiting examples include immunoglobulin domains, Fab, F(ab)'2, scFv, heavy-light chain dimers, immunoglobulin single variable domains, Nanobodies, domain antibodies, and single chain structures, such as a complete light chain or complete heavy chain.

Immunoglobulin variable domain

The term "immunoglobulin (Ig) domain", or more specifically "immunoglobulin variable domain" (abbreviated as "IVD") denotes an immunoglobulin domain essentially consisting of four "framework regions" which are referred to in the art and herein as: i) "framework region 1 " or "FR1 ", and ii) "framework region 2" or "FR2", and iii) "framework region 3" or "FR3", and iv) "framework region 4" or "FR4", respectively. These framework regions are interrupted by three "complementarity determining regions" or "CDRs", which are referred to in the art and herein as i) "complementarity determining region 1 " or "CDR1", and ii) "complementarity determining region 2" or "CDR2", and iii) "complementarity determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be indicated as follows: FR1 -CDR1 -FR2-CDR2-FR3-CDR3-FR4. Antigen specificity of an antibody is conferred by its IVDs, which comprise the antigen-binding site and determine the antibody’s ability to recognize and bind a particular epitope. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e. , a total of 6 CDRs will be involved in the formation of an antigen binding site. Given the aforementioned definition, the antigen-binding domain of a conventional 4-chain antibody (e.g., IgG, IgM, IgA, IgD, or IgE, as commonly understood) or its derivatives, such as a Fab fragment, a F(ab')2 fragment, an Fv fragment including disulfide-linked Fv or a scFv fragment, or a diabody (all recognized in the field), demonstrates affinity towards the corresponding epitope of an antigen through a pair of associated immunoglobulin domains, typically involving light and heavy chain variable domains, known as VH-VL pairs, which collectively recognize the epitope of the respective antigen.

Immunoglobulin single variable domain (ISVD)

An immunoglobulin single variable domain (ISVD) as used herein, refers to a protein with an amino acid sequence comprising 4 Framework regions (FRs) and 3 complementary determining regions (CDRs) according to the formula: FR1 -CDR1 -FR2-CDR2-FR3-CDR3-FR4. An "immunoglobulin domain" of this invention also refers to "immunoglobulin single variable domains" ("ISVDs"), equivalent to the term “single variable domains”, and defines molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from "conventional" immunoglobulins or their fragments, wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. The binding site of an immunoglobulin single variable domain is formed by a single VH/VHH or VL domain. Hence, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDR’s. As such, the single variable domain may comprise a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH or VHH sequence) or a suitable fragment thereof, provided it is capable of forming a single, functional antigen-binding unit. That is, the antigen-binding activity must be inherent to the single variable domain, such that it does not require pairing with another variable domain to confer antigen specificity.

In particular, the immunoglobulin single variable domain may be a Nanobody" (also referred to as "Nanobody", "nanobody" or "Nb"), or a suitable fragment thereof. Of note, Nanobody", Nanobodies" and Nanoclone" are registered trademarks of Ablynx N.V. (a Sanofi Company). For a general description of nanobodies, reference is made to the detailed explanation below and the prior art cited herein, such as to W02008/020079. "VHH domains", also known as VHHs, VHH domains, VHH antibody fragments, and VHH antibodies, have originally been described as the antigen binding immunoglobulin (Ig) (variable) domains of "heavy chain antibodies (HCAbs)" (i.e., of antibodies devoid of light chains)¹¹². These HCAbs, which are naturally present in camelids, lack the first domain of the constant region (CH1 ), which exists in the genome but is spliced out during mRNA processing. The antigen-binding site of HCAbs is composed of a single variable domain (i.e., a VHH), resembling the heavy chain variable domain (VH) of conventional antibodies, albeit with remarkable sequence differences at the second framework (FR2) and the third complementaritydetermining region (CDR3). The differences include amino acid substitutions at specific positions, such as V37F (Vai at position 37 in the VH to Phe in the VHH), or V37Y, G44E, L45R or L45C, and W47G (numbers refer to the amino acid positions numbered according to Kabat et al., 7997¹¹³). In the conventional VHs, these FR2 amino acids interact with the variable domain of the light chain (VL) and are conserved during evolution. The complementarity-determining region 3 (CDR3) of a VHH domain is, on average, longerthan that of a conventional VH domain and is often structurally stabilized by an interloop disulfide bond. These features enable HCAbs to recognize a broad spectrum of epitopes, including those that may not be accessible or recognizable by conventional antibodies with both heavy and light chains. As a result, HCAbs offer a versatile and valuable tool for various applications in research, diagnostics, and therapy, especially where traditional antibodies may face challenges in epitope recognition or accessibility. A high titer and a complex repertoire of HCAbs can be obtained from immunized or infected dromedaries or llamas¹¹⁴.

Following, the term "VHH domain" has been chosen to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VH domains") and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VL domains"). For a further description of VHHs and nanobodies, we refer to the review articles by Muyldermans ^{5 6}, as well as to the following patent documents: WO94/04678, WG95/04079 and WO96/34103 of the Vrije Universiteit Brussel; WO94/25591 , WO99/37681 , WG00/40968, WG00/43507, WG00/65057, WG01 /40310, WG01 /44301 , EP1134231 and WO2/48193 of Unilever; WO97/49805, WG01 /21817, WG03/035694, WG03/054016 and WG03/055527 of the Vlaams Instituut voor Biotech nologie (VI B); WG03/050531 of Algonomics N.V. and Ablynx N.V.; WG01/90190 by the National Research Council of Canada; WG03/025020 (EP1433793) by the Institute of Antibodies; as well as WG04/041867, WG04/041862, WG04/041865, WG04/041863, WG04/062551 , WG05/044858, WG06/40153, WO06/079372, WO06/122786, WO06/122787 and WO06/122825 by Ablynx N.V., and the further published patent applications by Ablynx N.V.

As described in these references, a nanobody/Nb (in particular VHH sequences and partially humanized nanobodies) can be characterized by the presence of one or more "hallmark residues" in one or more of the framework sequences. Further descriptions of nanobodies - including methods of humanization and/or camelization, as well as other modifications, fragments, derivatives, or "nanobody fusions", multivalent or multispecific constructs (with non-limiting examples of linker sequences), and various modifications designed to enhance nanobody half-life, along with methods of preparation - are provided in W02008/101985 and WO2008/142164, among other references. Nanobodies form the smallest antigen binding fragment that completely retains the binding affinity and specificity of a full-length antibody. Nanobodies possess exceptionally long complementarity-determining region 3 (CDR3) loops and a convex paratope, which allow them to penetrate into hidden cavities of target antigens.

Determination of CDR regions may be performed using different methods, such as the designation based on contact analysis and binding site topography as described in MacCallum etal., 7996¹¹⁷. Or alternatively, the annotation of CDRs may be done according to AbM (AbM is Oxford Molecular Ltd.'s antibody modelling package as described at http://www.bioinf.org.uk/abs/index.html¹¹⁸), Chothia¹¹⁹, Kabat^113,120, or IMGT¹²¹. These annotations further encompass the delineation of complementarity-determining regions (CDRs) and framework regions (FRs) within immunoglobulin-domain-containing proteins. Such annotations are based on well-established methodologies familiar to those skilled in the art, who can apply them to any immunoglobulin protein sequence without undue experimentation. While minor variations may exist between these annotation schemes, they consistently aim to identify the loop regions primarily responsible for antigen binding.

It should be noted that - as is well known in the art for VH domains and for VHH domains - the total number of amino acid residues in each of the CDRs may vary, so may not correspond to the total number of amino acid residues present in the sequence, or indicated by the numbering system applied. The Kabat numbering system, typically chosen for VHH amino acid numbering (also in this application, unless stated otherwise), for instance shows that one or more positions may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering. This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. The total number of amino acid residues in a VH domain and a VHH domain will usually be in the range of from 110 to 120, often between 112 and 115. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein. Furthermore, alternative VHH or antibody numbering systems may also apply, such as IMGT numbering for example.

VHHs or Nbs are often classified in different sequences families or even superfamilies, as to cluster the clonally related sequences derived from the same progenitor during B cell maturation¹²². This classification is often based on the CDR sequence of the Nbs, and wherein for instance each Nb family is defined as a cluster of (clonally) related sequences with a sequence identity threshold of the CDR3 region. Within a single VHH family defined herein, the CDR3 sequence is thus identical or very similar in amino acid composition, preferably with at least 80 % identity, or at least 85 % identity, or at least 90 % identity in the CDR3 sequence, resulting in Nbs of the same family binding to the same binding site, having the same effect or functional impact.

Immunoglobulin single variable domains, such as "domain antibodies" and "nanobodies" (including VHH domains), can be subjected to humanization. This process is aimed at increasing the degree of sequence identity with the closest human germline sequence¹²³. In particular, humanized immunoglobulin single variable domains, such as nanobodies (including VHH domains) may be immunoglobulin single variable domains in which at least one amino acid residue is present (and in particular, at least one framework residue) that is and/or that corresponds to a humanizing substitution (as defined herein). Potentially useful humanizing substitutions may be identified by comparing the framework region sequences of a naturally occurring VHH with those of one or more closely related human VH sequences. Based on this comparison, one or more candidate humanizing substitutions — or combinations thereof — can be selected and introduced into the VHH sequence using any suitable method known in the art, as further described herein. The resulting humanized VHH variants can then be evaluated for target-binding affinity, stability, expression yield and efficiency, and/or other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person. Based on the preceding description, the framework regions of an immunoglobulin single variable domain - such as a Nanobody, including VHH domains - may be partially or fully humanized.

Humanized immunoglobulin single variable domains, in particular Nanobody™, may have several advantages, such as a reduced immunogenicity, compared to the corresponding naturally occurring VHH domains. By "humanized" is meant that the variable domain, such as a Nanobody or VHH domain, has been mutated to reduce or eliminate immunogenicity when administered to human patients. In particular, humanization refers to the introduction of amino acid substitutions - typically within the framework regions - to make the sequence more similar to human immunoglobulin sequences, thereby minimizing the risk of eliciting an immune response. The humanizing substitutions should be chosen such that the resulting humanized amino acid sequence and/or VHH still retains the favorable properties of the VHH, such as the antigen-binding capacity. Based on the description provided herein, the skilled person will be able to select humanizing substitutions or suitable combinations of humanizing substitutions which optimize or achieve a desired or suitable balance between the favorable properties provided by the humanizing substitutions on the one hand and the favorable properties of naturally occurring VHH domains on the other hand. Such methods are known by the skilled addressee. A human consensus sequence can be used as target sequence for humanization, but also other means are known in the art. One alternative includes a method wherein a skilled person aligns a set of human germline alleles - such as, but not limited to, alleles of the IGHV3 family - to identify residues in a target sequence that are suitable for humanization. Alternatively, a subset of human germline alleles that are most homologous to the target sequence may be selected and aligned to serve as a reference for determining appropriate humanizing substitutions. Alternatively, the VHH sequence may be analyzed to identify its closest homolog among known human germline alleles, which is then used as a template for designing humanized constructs. A humanization technique applied to Camelidae VHHs may also be performed by a method comprising the replacement of specific amino acids, either alone or in combination. Such replacements may be selected based on prior literature, established humanization methodologies, human consensus sequences, or from the human germline alleles most similar to the natural VHH sequence of interest. As shown by the data on VHH entropy and variability presented in Tables A5-A8 of W008/020079, certain amino acid residues within the framework regions exhibit higher conservation between humans and Camelidae than others. Generally, although the invention in its broadest sense is not limited thereto, any substitutions, deletions or insertions are preferably made at positions that are less conserved. Also, generally, amino acid substitutions are preferred over amino acid deletions or insertions. For instance, a human-like class of Camelidae single domain antibodies contain the hydrophobic FR2 residues typically found in conventional antibodies of human origin or from other species, but compensating this loss in hydrophilicity by other substitutions at position 103 that substitutes the conserved tryptophan residue present in VH from double-chain antibodies. As such, peptides belonging to these two classes show a high amino acid sequence homology to human VH framework regions and said peptides might be administered to a human directly without expectation of an unwanted immune response therefrom, and without the burden of further humanization. Indeed, some Camelidae VHH sequences display a high sequence homology to human VH framework regions and therefore said VHH might be administered to patients directly without expectation of an immune response therefrom, and without the additional burden of humanization.

Suitable mutations, particularly substitutions, can be introduced during humanization to produce a polypeptide with reduced binding to pre-existing antibodies (see, for example, WO2012/175741 and WO2015/173325). Such substitutions may occur at one or more of the following positions: 11 , 13, 14, 15, 40, 41 , 42, 82, 82a, 82b, 83, 84, 85, 87, 88, 89, 103, or 108 (numbering according to Kabat), but also at other positions. The amino acid sequences and/or VHHs of the invention may be suitably humanized at any framework residue(s), such as at one or more hallmark residues (as defined herewith) or at one or more other framework residues (i.e. non-hallmark residues) or any suitable combination thereof. Depending on the host organism used to express the amino acid sequence, VHH, or polypeptide of the invention, such deletions and/or substitutions may also be designed to remove one or more sites for posttranslational modification (e.g., glycosylation sites), as would be within the capabilities of a person skilled in the art. Alternatively, substitutions or insertions may be designed so as to introduce one or more sites for attachment of functional groups (as described herein), for example to allow site-specific pegylation.

In some cases, at least one of the typical Camelidae hallmark residues with hydrophilic characteristics at position 37, 44, 45 and/or 47 is replaced (see W02008/020079, Table A03). Another example of humanization includes substitution of residues in FR1 , such as position 1 , 5, 11 , 14, 16, and/or 28; in FR3, such as positions 73, 74, 75, 76, 78, 79, 82b, 83, 84, 93 and/or 94; and in FR4, such as position 103, 104, 108 and/or 111 (see W02008/020079, Tables A05-A08; all numbering accordingto Kabat).

"Antigen-binding proteins" or "antigen-binding domains", as described herein, may be derived from an antibody or from alternative antigen-binding proteins with different folds. These alternative binding proteins include, but are not limited to, avimers, DARPins, alphabodies, affitins, nanofitins, anticalins, monobodies, and lipocalins.

Fc fusion

The term "Fc-fusion", as used herein, refers to the genetic fusion of one or more proteins or protein domains, such as antigen-binding fragments or antigen-binding domains, with an Fc constant domain, resulting in a dimeric structure that resembles an antibody when expressed in a recombinant host. Specifically, antibody fragments or single domain antibodies, such as ISVDs, may be fused at their C-terminus to the N-terminus of an Fc domain, preferably through a linker or hinge region. Alternatively, these antibody fragments or single domain antibodies, including ISVDs, may be fused at their N-terminus to the C-terminal end of an Fc domain or Fc tail, as used interchangeably herein, also preferably via a linker or hinge region. Such Fc-fusion constructs may include one or more VHHs or nanobodies, as described herein, and may combine the antigenbinding specificity of the single domain antibodies with the effector functions and extended halflife conferred by the Fc domain.

Panning

The term “panning” or “biopanning” refers to an affinity selection technique used to identify molecules that bind to a specific target. For example, biopanning can be used to identify and isolate peptides, proteins, or other molecules that bind to a particular target from a large library displayed on the surface of bacteriophages, yeast, or other systems. This process typically involves incubating the library with the target, washing away unbound molecules, eluting bound molecules, and amplifying them to create an enriched library. The cycle of selection and amplification may be repeated several times to increase the enrichment of high-affinity binders.

Medicament

The term “medicament” refers to any substance or combination of substances designated for treating, preventing, or diagnosing a disease, injury, or pathological condition in humans or nonhuman animals. This includes a wide range of formulations and delivery methods, such as oral medicaments, which can be i) biopharmaceuticals like insulin and monoclonal antibodies (e.g., adalimumab), ii) natural compounds like plant extracts (e.g., digitalis) and microbial metabolites (e.g., penicillin), iii) synthetic compounds like acetaminophen and ibuprofen, and iv) semisynthetic compounds like amoxicillin. Furthermore, topical medicaments encompass i) ointments (e.g., hydrocortisone cream), ii) creams and gels (e.g., clotrimazole), and iii) lotions (e.g., calamine lotion). Inhalable medicaments include i) aerosols and sprays (e.g., albuterol inhaler), and ii) nebulizers. Injectable medicaments encompass i) intravenous injections (e.g., vancomycin), and ii) intramuscular and subcutaneous injections (e.g., vaccines and insulin). Transdermal medicaments include adhesive patches (e.g., nicotine patches and fentanyl patches). Rectal, urethral and vaginal medicaments comprise suppositories (e.g., glycerin suppositories and miconazole). Ophthalmic and otic medicaments involve eye drops and ointments (e.g., ciprofloxacin), and ear drops (e.g., ofloxacin). Thus, the term “medicament” broadly encompasses various therapeutic agents designed for different routes of administration, each tailored to deliver specific therapeutic effects (such as pharmacologic effects) to treat, prevent, or diagnose diseases, injuries, or pathological conditions. Additionally, the term covers pharmaceutically acceptable salts, esters, solvates, and hydrates of pharmaceutically active substances, among other modifications used in the field. Treatment

The terms “treatment” “treating” and “treat” collectively denote any indication of success in therapy, alleviation, or prevention of an injury, disease, or pathological condition. This encompasses both objective and subjective parameters, such as reduction, remission, or alleviation of symptoms, and/or rendering the injury, disease, or pathological condition more manageable for the affected subject or individual. These terms also include decelerating the rate of progression, degeneration, and/or decline associated with an injury, disease, or pathological condition, mitigating the severity of the final degenerative outcome, and enhancing the affected individual's or subject's physical and/or mental well-being. The treatment or alleviation of symptoms may rely on objective or subjective criteria, including findings from diagnostic tests, physical examinations, neuropsychiatric assessments, and/or psychological evaluations. Moreover, the term “treating” and its variations may encompass preventive or prophylactic measures against an injury, disease, or pathological condition. In essence, the term “treatment,” as employed herein, encompasses any method aimed at curing, ameliorating, or preventing a disease, injury, or pathological state. Treatment interventions may serve to prevent the onset of the disease, impede its progression, alleviate its manifestations and/or symptoms, fully or partially eradicate the disease's underlying cause, reduce the duration of the disease, or achieve a combination of some or all of these objectives.

Disease

The term “disease” or “condition” or “disorder” refers to a pathological state that adversely affects the structure and/or function of an organism or its part, typically characterized by specific physical or psychological/mental symptoms and/or molecular or biochemical abnormalities. Diseases can arise from various causes, including genetic mutations, infections, environmental factors, or lifestyle choices, leading to disturbances in normal physiological processes. There are two primary categories of diseases: communicable and non-communicable. Communicable diseases, also known as infectious diseases, can be transmitted from one organism to another through various means, such as direct contact, airborne droplets, and/or contaminated surfaces. They typically involve infectious agents such as bacteria, viruses, fungi, or parasites. Examples of communicable diseases include influenza, tuberculosis, AIDS, malaria, and COVID-19. On the other hand, non- communicable diseases are conditions that cannot be transmitted from one organism to another. They often result from a complex interplay of genetic, environmental, and lifestyle factors. These conditions tend to develop over time and include cardiovascular diseases, neurodegenerative disorders like Alzheimer's and Parkinson's disease, psychiatric diseases, malignancies, and autoimmune conditions, among others. Treatment of a disease involves medical interventions designed to alleviate or eliminate symptoms, manage complications, and/or target underlying causes to restore health and well-being in an affected organism.

Autoimmune disease

The terms “autoimmune disease”, “autoimmune condition”, “autoimmune disorder”, “autoreactive disease”, “autoreactive condition” and “autoreactive disorder” all refer to a group of chronic ailments that develop in humans when the immune system attacks the host's organs, tissues, or cells. This immune response manifests as inflammation, which may lead to tissue damage and organ failure. Autoimmune responses bear similarity to conventional immune reactions against pathogens, as they are prompted by the presence of specific antigens. However, in autoimmune responses, these antigens are “self-antigens” or “autoantigens”, i.e., molecules or substances naturally present in cells or tissues of an affected individual, which are mistakenly identified as foreign or harmful; this misidentification leads to an immune response against the body's own components. Autoantigens can encompass diverse molecular constituents, including peptides, proteins, carbohydrates, nucleic acids, and complexes and/or fragments thereof. For example, p53 has been described as an autoantigen in several autoimmune diseases, including lupus and scleroderma, TPO (thyroid peroxidase) is a major autoantigen in autoimmune thyroid diseases such as Hashimoto's thyroiditis, whereas antibodies targeting glutamic acid decarboxylase (GAD) are distinctive markers associated with Moersch-Woltman syndrome (stiffperson syndrome).

Autoreactive B cells play a crucial role in driving pathogenic processes in autoimmune diseases through their production of autoantibodies, secretion of cytokines, and presentation of autoantigens to T cells¹²⁴. On the other hand, self-reactive T cells, particularly CD4⁺ T cells, are implicated in mediating various aspects of autoimmune inflammation¹²⁵; in some diseases, they may function as effector cells directly involved in the killing of cells expressing their target autoantigens. In this context, “autoreactivity” is reflected in the affinity between the T cell receptor (TCR) and self-antigens, as well as the concentration of self-antigens in the tissues¹²⁶.

Numerous autoimmune diseases in humans are linked to variations or mutations in the human leukocyte antigen (HLA) locus¹²⁷. This supports the notion that T cells play a major role in the development of these disorders. To become autoreactive, T cells need to be activated by three types of signals provided by antigen-presenting cells (APCs), such as dendritic cells (DCs). These signals encompass: i) antigen-specific stimulation of the TCR via recognition of specific peptide- major histocompatibility complex (MHC) multimers on APCs; ii) ligand-mediated activation of one or more co-stimulatory receptors (such as ICOS, CD28, CD40 and 0X-40) promoting T cell differentiation and proliferation; and iii) stimulation with cytokines^{128 129}.

On the other hand, co-inhibitory receptors (such as PD-1 , TIM-3, TIGIT, CTLA-4 and LAG-3) transduce signals that counteract T cell activation and suppress immune responses against (auto)antigens¹³⁰. Notably, the expression of co-inhibitory receptors is often induced uponT cell costimulation; this hints at the presence of a negative feedback loop that finely tunes immune system activation under physiological conditions¹³¹. Indeed, excessive co-stimulation and/or inadequate co-inhibition result in aberrant? cell activation, potentially causing a breakdown of self-tolerance by stimulating and expanding autoreactive T cells¹³⁰. In addition to T cells, co-stimulatory and co- inhibitory receptors and their ligands (collectively termed “immune checkpoints”, as defined in this application) also play a role in regulating immune responses of natural killer (NK) cells, innate lymphoid cells, and myeloid cells¹³².

Autoreactivity associated with autoimmune conditions, as referred to herein, stems from various maladaptive molecular mechanisms, including: i) insufficient central tolerance (self-reactive lymphocytes evade deletion in the thymus and enter the peripheral circulation); ii) defective peripheral tolerance (peripheral mechanisms like anergy, regulatory? cells, and immune privilege fail to suppress self-reactive lymphocytes); iii) molecular mimicry (auto-antigens structurally resemble pathogenic antigens, leading to immune responses due to cross-reactivity); iv) epitope spreading (initial immune responses expose additional epitopes, triggering an autoimmune reaction); v) genetic susceptibility (specific genetic factors, such as HLA alleles, increase the risk of autoimmune responses); and/or vi) environmental triggers (external factors like pathogens, toxins, or UV radiation provoke or exacerbate autoimmune responses).

Autoimmune conditions as referred to herein can affect any organ system in any individual, although women are up to four times more likely to develop these diseases compared to men¹³³. Despite common underlying mechanisms, the clinical presentations of autoimmunity are markedly diverse; the manifestations span from subtle abnormalities that evade diagnosis/detection to acute, potentially life-threatening organ dysfunction³. Indeed, autoreactivity exists on a spectrum, ranging from a basal physiological level essential for lymphocyte selection and the maintenance of immune system homeostasis⁵, through an intermediate level characterized by the presence of circulating autoantibodies and immune tissue infiltrates unassociated with clinical symptoms, to pathogenic autoimmunity linked with immune-mediated dysfunction and/or tissue damage¹³⁴. In this line, symptoms of autoimmune diseases often display considerable variation, largely depending on the specific type of condition and the location within the body that is affected. However, these symptoms commonly appear intermittently and can vary widely in their severity.

The most prevalent human autoimmune diseases include: Addison’s disease, ankylosing spondylitis, coeliac disease, childhood-onset type 1 diabetes, Graves’ disease, Hashimoto’s thyroiditis, inflammatory bowel disease (Crohn’s disease or ulcerative colitis), multiple sclerosis, myasthenia gravis, pernicious anemia, polymyalgia rheumatica, primary biliary cholangitis, psoriasis, rheumatoid arthritis (including its specific subtypes, such as Still disease, Caplan syndrome, rheumatoid spondylitis, and others), Sjogren’s syndrome, systemic lupus erythematosus, systemic sclerosis, vasculitis, and vitiligo¹. Notably, the list of autoimmune diseases provided above is not exhaustive.

Graft versus host disease

The term "graft versus host disease" (GVHD) refers to a complex immunological disorder that occurs when immunocompetent donor-derived immune cells, primarily T lymphocytes, recognize the recipient’s tissues as foreign following allogeneic hematopoietic stem cell transplantation (HSCT) or organ transplantation. This results in an immune-mediated attack against the host’s cells, leading to tissue inflammation and damage. GVHD is typically classified into acute and chronic forms, characterized by distinct clinical features a nd pathophysiological mechanisms. The disease process involves donor T cell activation by host antigen-presenting cells presenting alloantigens, resulting in cytokine release, recruitment of effector cells, and destruction of target organs such as the skin, liver, gastrointestinal tract, and lungs. GVHD severity depends on multiple factors including donor-recipient histocompatibility, conditioning regimens, and/or immunosuppressive therapies.

Allergic disease

The term "allergic disease" refers to a group of immune-mediated disorders characterized by hypersensitive or inappropriate immune responses to otherwise harmless environmental antigens known as allergens. These diseases involve the activation of the immune system, particularly the production of allergen-specific IgE antibodies, sensitization of mast cells and basophils, and subsequent release of inflammatory mediators upon allergen re-exposure. Allergic diseases encompass conditions such as allergic rhinitis, asthma, atopic dermatitis, food allergies, and anaphylaxis. The pathophysiology typically involves a type I hypersensitivity reaction, with Th2- skewed immune responses, eosinophilic inflammation, and cytokine production (e.g., IL-4, IL-5, IL- 13). Clinical manifestations vary depending on the target organ system but generally include inflammation, tissue remodeling, and impaired function resulting from immune activation against innocuous antigens.

Allergic diseases are systemic disorders arising from dysfunction of the immune system. Conditions such as allergic rhinitis (AR), allergic asthma syndrome (AAS), atopic dermatitis (AD), food allergy (FA), and eczema resultfrom complex interactions between genetic predisposition and environmental triggers. The World Health Organization (WHO) lists allergic diseases among the top three conditions requiring prevention and control in the 21st century. While allergic diseases are systemic in nature, they frequently present as localized symptoms that may escalate to anaphylactic shock in severe cases. Their prevalence is high and continues to rise, causing significant distress and burden to affected individuals. Globally, it is estimated that AR and AAS affect approximately 500 million and 300 million individuals, respectively. Mortality rates associated with AAS are 90 per million in women and 170 per million in men, with about 96% of asthma-related deaths occurring in low- and middle-income countries. Food allergies affect an estimated 1-10% of the global population. The prevalence of AD stands at approximately 8%, with lifetime incidence reaching up to 20%. In 2019 alone, over 171 million people worldwide were diagnosed with AD. Due to the different anatomical sites affected, clinical and pathological manifestations of allergic diseases vary. In AR, exposure to airborne allergens- such as dust mite fecal particles, cockroach debris, pet dander, molds, and pollens - triggers an immune response characterized by infiltration of inflammatory cells (e.g., mast cells, CD4⁺ T cells, B cells, macrophages, and eosinophils) into the nasal mucosa. Traditionally, Th2 cells have been associated with the production of immunoglobulin E (IgE) and cytokines like IL-3, IL-4, IL-5, and IL- 13. However, recent findings suggest that follicular helper T (Tfh) cells play a more critical role in regulating IgE production. Cross-linking of allergen-bound IgE on mast cells results in the release of histamine and leukotrienes, causing vasodilation, increased vascular permeability, mucus secretion, pruritus, and smooth muscle contraction. A secondary wave of inflammation follows within 4-8 hours, contributing to symptoms such as persistent nasal congestion. AR and AAS share similar immunopathological features, including infiltration by eosinophils, mast cells, and Th2 cells. Although airway remodeling is a well-known feature in AAS, evidence suggests that similar changes may also occur in AR. AAS-specific alterations include epithelial hyperplasia, goblet cell metaplasia, and increased mucus production. Submucosal changes - such as smooth muscle hypertrophy and collagen deposition — lead to airway narrowing and contribute to classic asthma symptoms like wheezing, coughing, and breathlessness. In AD, pathogenesis involves a multifaceted interplay between epidermal barrier dysfunction, microbial imbalance, and dysregulated type 2 T cell responses. This leads to weakened skin integrity, susceptibility to Staphylococcus aureus colonization, and localized inflammation characterized by epidermal edema and immune cell infiltration. Th2 cytokines further impair barrier function and exacerbate microbial dysbiosis, perpetuating chronic itch and inflammation. Food allergies represent IgE- mediated type I hypersensitivity reactions. In the sensitization phase, initial exposure to a food allergen disrupts immune tolerance, leading to the production of specific IgE and release of mediators such as histamine and platelet-activating factor. Upon re-exposure, degranulation of mast cells and basophils triggers acute gastrointestinal symptoms and, in severe cases, systemic anaphylaxis. Persistent exposure may cause chronic inflammation and mast cell accumulation, contributing to ongoing gastrointestinal distress. The pathogenesis of allergic diseases is multifactorial, involving genetic, epigenetic, environmental, microbial, and immunological components. Their tendency to recur frequently not only compromises quality of life but also imposes a substantial financial burden on healthcare systems and patients alike. This manuscript aims to offer a comprehensive overview of allergic diseases -from their historical context and underlying mechanisms to current treatment approaches - serving as a valuable reference for clinicians and researchers in the field.

Nucleic acid

The term "nucleic acid" refers to a class of biopolymers comprising nucleotide monomers, which may be single-stranded or double-stranded, linear or circular. Nucleic acids serve as carriers of genetic information and mediators of essential cellular functions. The term encompasses deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and their analogs and/or derivatives, including chemically modified forms.

DNA is typically a double-stranded molecule composed of nucleotides containing the bases adenine (A), thymine (T), cytosine (C), and guanine (G), and it serves as the primary medium for long-term storage and transmission of genetic information in cells. RNA is most often singlestranded and comprises nucleotides containing adenine (A), uracil (U), cytosine (C), and guanine (G). RNA molecules are involved in a wide range of cellular functions, particularly in gene expression and regulation. RNA species include, but are not limited to: (i) messenger RNA (mRNA), which carries coding information for protein synthesis; (ii) transfer RNA (tRNA) and ribosomal RNA (rRNA), which are involved in protein translation; (iii) small interfering RNA (siRNA) and microRNA (miRNA), which mediate post-transcriptional gene silencing; (iv) long non-coding RNA (IncRNA), which contributes to chromatin remodeling, transcriptional regulation, and other epigenetic processes; (v) circular RNA (circRNA), which may function as regulatory molecules or miRNA sponges (sequesters). The term "nucleic acid" also encompasses synthetic and modified nucleic acids, such as peptide nucleic acids (PNAs), locked nucleic acids (LNAs), morpholino oligomers, and aptamers, as well as chemically modified oligonucleotides designed to enhance the stability, affinity, or resistance to degradation of parental molecules. These variants are widely used in biotechnology, diagnostics, therapeutics, and gene editing. Nucleic acids are central to the processes of gene replication, transcription, and translation, and are fundamental to genetic engineering, molecular biology, and synthetic biology applications.

Vector

The terms "vector", "vector construct", "expression vector", "recombinant vector", or "gene transfer vector", as used herein, refer to a nucleic acid molecule capable of carrying and delivering another nucleic acid molecule to which it is operably linked. A vector may include any nucleic acid-based construct known to those skilled in the art, including, but not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors such as lambda phage, viral vectors including lentiviral, adenoviral, adeno-associated viral (AAV), and baculoviral vectors, and artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), and P1 -derived artificial chromosomes (PAC). Such vectors may function as cloning or expression vectors, or as delivery vehicles suitable for transferring genetic material into target cells or organisms. In certain embodiments, viral vectors, including lentiviral and adenoviral vectors, are utilized to facilitate efficient nucleic acid delivery. Expression vectors may be either plasmid-based or viral in nature and typically comprise a nucleic acid sequence encoding a desired protein, operably linked to one or more regulatory sequences required for transcription and, where applicable, translation of the encoded protein in a chosen host organism. Such host systems may include prokaryotic or eukaryotic cells, including bacterial, yeast, plant, insect, or mammalian cells, or may include in vitro expression systems. Cloning vectors, in contrast, are primarily used for the manipulation, amplification, and subcloning of nucleic acid sequences and may not contain the necessary elements for expression. Construction of suitable vectors is well known in the field and can be achieved using standard recombinant DNA techniques, such as those described in: i) "Molecular Cloning: A Laboratory Manual", 4^th Edition (2012), by SambrookJ and Green M³¹; ii) "Gene Transfer and Expression Protocols", Ed. Murray EJ, The Humana Press, Clifton, NJ¹³⁵; or iii) "Gene Therapy Protocols", Ed. Le Doux JM, The Humana Press, Clifton, NJ¹³⁶. Nucleic acid molecules, vectors, or related constructs encoding polypeptides as described herein can be utilized in therapeutic applications, including but not limited to gene therapy. The term "gene therapy", as used herein, refers to the administration of a nucleic acid molecule, such as DNA or RNA, to a subject, wherein said molecule is capable of being expressed in vivo and thereby producing a therapeutic effect. Vectors used in gene therapy may deliver the desired gene to target cells and may include viral vectors such as adeno-associated virus (AAV), adenovirus, or lentivirus, capable of inducing transient or stable gene expression. Gene therapy approaches may be implemented using methods including viral vector delivery, direct injection of plasmid DNA, biolistic delivery of naked nucleic acid (e.g., "gene gun"), electroporation, liposomal encapsulation, or nanoparticle-mediated delivery. Additionally, gene transfer may be performed using artificial exosomes or lipid-based carriers designed to protect nucleic acids from degradation and enhance uptake by target cells. Administration may be performed via systemic or localized routes, including but not limited to intravenous, intramuscular, subcutaneous, intraperitoneal, intrathecal, or intracerebroventricular delivery, depending on the therapeutic context.

Pharmaceutical composition

In the context of the present invention, the term "pharmaceutical composition" or "medicinal composition" pertains to a mixture comprising one or more specific substances aimed at providing pharmacological activity or exerting a direct effect in the cure, mitigation, treatment, and/or prevention of a disease or pathological condition. A "pharmaceutical composition" administered to a subject/individual may directly influence the restoration, correction, or modification of physiological functions in said subject/individual. Accordingly, a pharmaceutical or medicinal composition refers to any formulation comprising an active ingredient - such as the allosteric modulator of an immunoinhibitory complex, as disclosed herein - either alone, in dispersion, or as part of a composite, and optionally one or more additional active ingredients and/or pharmaceutically acceptable carriers, excipients, diluents or adjuvants.

The term "pharmaceutically acceptable" refers to a material suitable for administration to an individual or subject, which, upon contact with the tissues of the individual or subject, does not cause unacceptable toxicity, irritation, allergic reaction, or other adverse biological effects, and exhibits a benefit-to-risk profile appropriate for the intended therapeutic application. In other words, such a material may be safely administered together with an active ingredient as part of a pharmaceutical composition without causing undesirable biological effects or negatively interfering with the therapeutic activity of the active ingredient or other components of the composition. The terms "carrier", "excipient", "diluent" and "adjuvant", and in particular the terms "pharmaceutically acceptable carrier", "pharmaceutically acceptable excipient", "pharmaceutically acceptable diluent" and "pharmaceutically acceptable adjuvant", refer to substances that facilitate the formulation, delivery, stability, and/or efficacy of a therapeutically active ingredient of a pharmaceutical composition, while being non-toxic, physiologically compatible, and free from inducing harmful immune or toxicological responses when administered to an individual/subject alone at relevant dosages. A "carrier" refers to any substance that serves as a medium or vehicle to deliver an active ingredient to a subject. Carriers may protect the active ingredient, enhance its bioavailability, prolong its circulation time, or assist in targeting specific tissues. Suitable carriers include, but are not limited to, liposomes, polymeric matrices, nanoparticles such as lipid nanoparticle (LNPs), microspheres, proteins such as albumin, biodegradable polymers such as polylactic acid, polyglycolic acid, and their copolymers, as well as inactivated viral particles. An "excipient" is any pharmaceutically inactive substance included in a pharmaceutical composition to aid in the manufacturing process, improve stability, enhance taste, facilitate administration, or contribute to the physical properties of the dosage form. Excipients can include binders, fillers, disintegrants, preservatives, buffering agents, flavoring agents, colorants, surfactants, stabilizers, lubricants, and/or coatings. Examples of excipients are lactose, microcrystalline cellulose, mannitol, sorbitol, and various surfactants. While excipients have no direct therapeutic effect, they may be essential to the preparation of safe and effective pharmaceutical compositions. A "diluent" is a specific type of excipient used to dilute the active ingredient to a desired concentration or volume. Diluents are often liquid vehicles such as sterile water, saline, phosphate-buffered saline, glycerol, or ethanol, but may also be solid substances used in tablets or capsules. Diluents may contain auxiliary agents such as preservatives, wetting agents, emulsifiers, or buffering substances to maintain stability and compatibility. An "adjuvant" is a substance included in pharmaceutical or vaccine formulations to enhance or modulate the immune response to an antigen. Unlike carriers or excipients, adjuvants often have biological activity and can stimulate immune receptors or pathways to improve the magnitude, duration, or quality of the immune response. Suitable adjuvants include aluminum salts (such as aluminum hydroxide or aluminum phosphate), saponins, Toll-like receptor agonists, oil-in-water emulsions, and certain biodegradable polymers. A pharmaceutically acceptable adjuvant is one that enhances or modulates the immune response to an antigen without causing harmful side effects or excessive inflammation. Together, pharmaceutically acceptable carriers, excipients, diluents, and/or adjuvants facilitate the effective delivery, stability, and therapeutic efficacy of active ingredients in pharmaceutical compositions, while maintaining the overall safety and biocompatibility of the formulation. These components may function synergistically to optimize the performance of the active ingredient of the pharmaceutical formulation without compromising its intended biological/pharmacological/therapeutic activity or introducing adverse effects.

A pharmaceutically effective amount of polypeptides of the invention, together with a pharmaceutically acceptable carrier, excipient, diluent and/or adjuvant, is preferably an amount sufficient to produce a desired pharmacological/therapeutic effect or influence on the specific condition being treated. For therapeutic applications, the pharmaceutical composition of the invention may be administered to a patient using standard or approved methods of administration. Said administration may be carried out via any medically acceptable route, including, but not limited to, oral, parenteral (e.g., intravenous, intramuscular, subcutaneous, or intradermal), topical, transdermal, nasal, buccal, sublingual, pulmonary (e.g., via inhalation or aerosol), ophthalmic, otic, rectal, vaginal, urethral, intraperitoneal, intracardiac, intranasal, intraarticular, intrapleural, intravesical, intratumoral, intracerebral, intrathecal, intracerebroventricular, or by implantation of a depot or sustained-release formulation. The composition may be delivered using conventional systems or advanced delivery platforms, including nanoparticle-based carriers, liposomes, micelles, hydrogels, biodegradable polymer matrices, micro- or nano-encapsulation, viral or non-viral vectors, electroporation, microneedles, and infusion pumps. Additional formulation and delivery technologies, such as controlled-release systems, targeted delivery systems, inhalable dry powders, pressurized metered dose inhalers (pMDIs), nebulizers, nasal sprays, oropharyngeal sprays, and other site-specific or systemic delivery mechanisms, are also included within the scope of the present invention. The dosage and frequency of administration will depend on a variety of clinical and patient-specific factors, including, but not limited to, (i) the age, weight, sex, and general health condition of the patient; (ii) the severity and nature of the disorder beingtreated; (iii) the pharmacokinetics and pharmacodynamics of the active agent; (iv) the chosen route of administration; (v) the administration of other medications; (vi) the presence of individual contraindications; and (vi) the discretion of the attending physician, clinician or healthcare provider. The precise regimen may be determined based on therapeutic objectives and patient response.

The pharmaceutical composition described herein, optionally in combination with additional therapeutic agents, may be prepared in a variety of formulations. These include, but are not limited to: solutions, suspensions, emulsions, drops, tablets, pills, pellets, capsules (includingsoftor hard gelatin capsules and capsules containing liquid or solid fill), powders, sustained-release or controlled-release formulations, suppositories, aerosols, sprays, lyophilized powders, frozen suspensions, desiccated powders, or any other pharmaceutically acceptable form appropriate for the intended route of administration.

The polypeptide of the disclosure, in particular the allosteric modulator capable of binding and inducing positive cooperativity within an immunoinhibitory protein complex, may form a pharmaceutically acceptable salt. A "pharmaceutically acceptable salt" of the polypeptide disclosed herewith denotes a compound that possesses the desired pharmacological activity of the parent compound and includes either: i) an acid addition salt, formed with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with an organic acid such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4- toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]oct-2-ene-1 -yl carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, 3-hydroxy-2-naphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or ii) a salt formed when an acidic proton present in the parent compound is either replaced by an inorganic ion (e.g., an alkali metal ion such as Na⁺, K+ or Li⁺, an alkaline earth ion such as Ca2⁺ or Mg²⁺, an aluminum ion, or an ammonium ion) or coordinates with an organic base (e.g., ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, morpholine, piperidine, dimethylamine, diethylamine, and the like).

Detailed description

The present invention originates from research efforts aimed at developing novel strategies and tools for modulating signaling pathways of inhibitory immune receptor-ligand complexes. Specifically, the invention pertains to allosteric modulators that, upon binding to epitopes located on immune complexes formed between an inhibitory immune receptor and its ligand, induce positive cooperativity within these complexes, as defined herewith. The capacity of the allosteric modulators of this disclosure to induce positive cooperativity within inhibitory immune receptorligand complexes upon specific binding represents the key concept of the present disclosure. This modulatory activity reinforces and supports naturally occurring receptor-ligand interactions, thereby enabling controlled augmentation of inhibitory immune signaling in a (i) targeted, (ii) spatiotemporally selective, and (iii) endogenous ligand-dependent manner. This selective modulation offers a promising therapeutic approach for the treatment of inflammatory conditions, including but not limited to autoimmune diseases. The disclosure also relates to methodologies for identifying, selecting, and generating said allosteric modulators, as described herein.

In a first aspect, the invention provides novel modulators that are capable of allosterically binding to epitopes located on immunoinhibitory receptor-ligand complexes. Said modulators comprise an immunoglobulin single variable domain (ISVD), as defined herewith in detail, with an antigenbinding site specifically targeting the receptor-ligand complex. The receptor-ligand complex targeted by the allosteric modulator of this disclosure comprises at least one inhibitory surface immune receptor, as defined herewith, and at least one orthosteric ligand of that receptor, as defined herewith, wherein the components of the complex are typically associated through non- covalent interactions. Such a complex is referred to as an “immunoinhibitory complex” throughout this disclosure. Non-limiting examples of immunoinhibitory complexes targeted by the allosteric modulators of this invention include PD-1 »PD-L1 , PD-1 »PD-L2, CD200R»CD200L, TIGIT’PVR, VISTA»VSIG-3, CD16OHVEM, BTLA’HVEM, BTLA»HVEM»CD160, CTLA-4»B7-1 , TIM-3»Galectin- 9»Ceacam-1 , LAIR-1 •C'l q, and KIR3DL3»HHLA2, as well as other complexes made by the components as listed in Figure 1 and 2, and/or as provided in Figure 3.

Upon binding to the allosteric modulator disclosed herein, an immunoinhibitory complex exhibits increased affinity between its interacting subunits, in particular between the receptor and the ligand. This enhanced affinity results from positive cooperativity induced by conformational changes triggered by the binding of the allosteric modulator, which lowers the free energy of receptor-ligand interaction and thereby stabilizes the complexed state, as described in detail herein. Accordingly, the present disclosure applies the cooperativity framework to describe the interdependent behavior of binding sites within immunoinhibitory complexes upon interaction with the allosteric modulators disclosed herein.

The antigen-binding site of the ISVD of the allosteric modulator of this invention specifically binds an epitope that is predominantly present on an immunoinhibitory complex. This means that the epitope may not be exclusively present on the complex, although it can be exclusively present on said complex. The term ‘predominantly present’ used herein refers to the fact that the allosteric modulator specifically binds an epitope that is unique or exclusively present when the protein complex is formed, i.e. a complex-specific conformational epitope, though with the possibility that the allosteric modulator or binder retains at least partial binding capacity to the individual nonbound protein partners of said complex due to an at least partial presence of a connecting epitope, or due to the presence of a conformation of said individual protein that exposes at least partially the complex-specific conformational epitope. In such cases the allosteric modulator will predominantly bind the complex, as the optimal exposure of the epitope is present on the complex, but may also provide for residual binding, typically to a much lower extent, for the individual protein partners. The predominant presence of the epitope on the complex may thus also be referred to as a unique true complete binding site on the complex, and a weaker or partial binding site exposed on the individual protein(s) of said complex. The predominant presence of the epitope on the complex is thus considered as the optimal or stabilizing binding site of the allosteric modulator to provide fora PAM. In various embodiments, the epitope to which the allosteric modulator disclosed herein binds may be: (i) present exclusively on the receptor component of the immunoinhibitory complex; or (ii) present exclusively on the ligand component of the immunoinhibitory complex; or (iii) formed by residues contributed jointly by both the receptor and the ligand of the immunoinhibitory complex; or (iv) present on multiple copies of the receptor and/or ligand in the case of multimeric or oligomeric complexes (e.g., complexes comprising homodimers, heterodimers, or higher-order multimers, as described below in detail). In some embodiments, the epitope may correspond to a conformational determinant that only arises upon complexation of the receptor and ligand, such that it is absent or structurally distinct in the unbound (monomeric) forms of the individual protomers, such as individual hCTLA-4 or individual hB7.1. In other embodiments, the epitope may partially overlap with amino acid residues and/or structural features present in the uncomplexed receptor and/or ligand, but adopts a distinct conformation, spatial arrangement, and/or accessibility in the context of the complex, thereby influencing the binding affinity of the allosteric modulator. In some embodiments, fragments or partial forms of the epitope - comprising, for example, a subset of key amino acid residues critical for allosteric modulator recognition - may be present, to some extent, on the unbound receptor and/or ligand. These partial or incomplete epitope structures may exist in a conformationally disordered, sterically occluded, or otherwise non-native state, such that their affinity for the allosteric modulator is substantially reduced compared to the fully assembled immunoinhibitory complex. In such cases, the modulator may still engage with these cryptic epitopes, but typically with a decreased binding strength, altered kinetics, or lower functional potency. As used herein, the term "cryptic epitope" refers to a structural motif or molecular surface that is not readily accessible, properly conformed, or functionally competent for high-affinity modulator binding in the context of an uncomplexed receptor or ligand. These cryptic epitopes may be masked by intramolecular interactions, buried within the protein core, obscured by glycosylation, or otherwise rendered inaccessible under native conditions. Upon receptor-ligand complexation, such regions may undergo conformational rearrangement, allosteric unmasking, or spatial reorientation, thereby becoming exposed or structurally stabilized in a manner that permits functional interaction with the allosteric modulator of this disclosure. To exemplify this further, the cryptic epitope may correspond to a loop region or flexible domain in the receptor that adopts a defined conformation only upon ligand engagement. In another example, a binding pocket or groove formed at the receptor-ligand interface may be absent in either protomer alone, but is formed or stabilized upon complex formation. Alternatively, the epitope may be buried within a hydrophobic core in the unbound protein but becomes solvent-exposed as a result of complex-induced unfolding or domain swapping. Such differential binding may confer selectivity, wherein the modulator of the present disclosure preferentially associates with the receptor-ligand complexed state, thereby predominantly exerting its modulatory activity upon binding to the immunoinhibitory complex rather than to the unbound receptor or ligand protomers.

It is to be understood that the allosteric modulator described herein may target any type of immunoinhibitory complex, irrespective of the number or identity of its constituent subunits. Thus, the term "immunoinhibitory complex" refers to any assembly comprising one or more inhibitory surface immune receptor molecules, as defined herein, bound to one or more immune receptor ligand molecules, as also defined herein. Such complexes may be binary, ternary, quaternary, or higher-order multimeric assemblies and may involve homo- or hetero-multimeric interactions among receptors, ligands, or both. In one embodiment, the immunoinhibitory complex comprises a binary interaction between one immune receptor and one ligand. Non-limiting examples of such binary complexes include PD-1 »PD-L1 , PD-1 »PD-L2, BTLA»HVEM, and TNFR2»membrane-bound TNFa (mTNFa). In another embodiment, the immunoinhibitory complex comprises an assembly of two immune receptor molecules and one ligand molecule. Non-limiting examples include BTLA»HVEM»CD160 and Nrp-1 »Plexin-A4»B7-H4. In yet another embodiment, the immunoinhibitory complex comprises one immune receptor molecule interacting simultaneously with two distinct ligand molecules. Illustrative examples include PD-1 •Galectin-9»BTN1 A1 and Nrp-1 •B7-H4»Sema3A. In a further embodiment, the immunoinhibitory complex comprises two immune receptor molecules and two ligand molecules. An exemplary quaternary complex includes Nrp-1 •Plexin-A4»B7-H4»Sema6C. These complexes may form through sequential or cooperative binding and may adopt defined three-dimensional (3D) architectures that are suitable targets for allosteric modulation by the modulators disclosed herein. It shall also be understood that the immunoinhibitory complex to which the allosteric modulator of the present invention binds may comprise identical or non-identical receptor and/or ligand subunits. For example, in one embodiment, the immunoinhibitory complextargeted by the allosteric modulatorof this disclosure may comprise two CTLA-4 molecules and two B7-1 molecules, i.e . , CTLA-4* CTLA-4 »B7-1 *B7-1 , or two CTLA-4 molecules and two B7-2 molecules, i.e., CTLA-4*CTLA-4*B7-2*B7-2. In such higher- order assemblies, epitope presentation may be dependent on quaternary structure, intermolecular interface geometry, and/or spatial organization of domains, thereby enabling selective binding by the allosteric modulator. In certain embodiments, the immunoinhibitory complex may also include accessory molecules that modulate the activity, localization, or conformation of the receptorligand assembly. Such accessory molecules may include, but are not limited to, scaffold proteins, co-receptors, membrane tethers, signal-transducing adaptors, and chaperone proteins.

In various embodiments, the invention provides allosteric modulators that target transient immunoinhibitory complexes. As used herein, the terms "transient complex" and "transient protein- protein interaction" refer to non-permanent, reversible assemblies formed by inhibitory surface immune receptors and their respective ligands. These complexes are capable of dynamically associating and dissociating under physiological conditions, and each constituent protein is capable of existing stably in the unbound form. Transient protein- protein interactions are typically characterized by moderate to low binding affinities and short-lived interaction durations, often regulated by the spatial and temporal context of the cellular microenvironment. Factors influencing such transient interactions include, but are not limited to, receptor and ligand expression levels, subcellular localization, membrane compartmentalization, immune cell activation status, cytokine and/or hormonal signaling, and/or the presence of disease-associated stimuli such as inflammation. Byway of example, PD-1 forms transient complexes with its ligands, PD-L1 and PD-L2, duringthe initiation and regulation of immune responses^{137 138}. These interactions typically occur at the immunological synapse and serve to inhibit T cell receptor (TCR)-mediated signal transduction and dampen co-stimulatory signaling, thereby promoting immune tolerance and limiting excessive immune activation. The PD-1 »PD-L1 and PD-1 »PD-L2 interactions are dynamically regulated and are reversible, enabling precise control of T cell activity during physiological and pathological immune events.

In one embodiment, the allosteric modulator of the present invention selectively binds to an epitope that is predominantly present on the PD-1 »PD-L1 complex, but not on the PD-1 »PD-L2 complex. In a separate embodiment, the allosteric modulator selectively binds to an epitope that is predominantly present on the PD-1 »PD-L2 complex, but not on the PD-1 »PD-L1 complex. Without intendingto be limited bytheory, it is noted that although PD-L1 and PD-L2 are both ligands of PD-1 , they engage PD-1 via partially overlapping but structurally distinct interaction surfaces, resulting in conformationally unique transient complexes¹³⁹. Thus, the allosteric modulators targeting the PD-1 »PD-L2 complex are structurally and functionally distinct from those targeting the PD-1 »PD-L1 complex, and both types of modulators may exhibit different binding affinities toward their respective target complexes. The molecular distinction between the PD-1 »PD-L1 and PD-1 »PD-L2 complexes may arise from differences in the receptor-ligand interface, the spatial orientation or flexibility of interacting domains, epitope accessibility, and/or conformational dynamics induced upon complex formation, among others. These differences may yield structurally unique composite epitopes that are selectively recognized by the allosteric modulators of this disclosure. Accordingly, allosteric modulators capable of differentiating between these complexes are within the scope of the present invention. Examples 18 and 19 are provided herewith to illustrate this principle. Moreover, in various embodiments, the allosteric modulator of the present invention may bind and modulate immunoinhibitory complexes comprising subunits/protomers derived from any organism, without limitation to species, phylum, or kingdom of origin. Such subunits/protomers may include immune receptors, ligands, or accessory proteins that are derived from either: (i) protostomes, including but not limited to arthropods (e.g., insects, crustaceans), mollusks, flatworms, annelids, and nematodes; or(ii) deuterostomes, including but not limited to vertebrates such as fish, amphibians, reptiles, birds, and mammals. In certain embodiments, the protein subunits of the complex bound by the allosteric modulator of the present invention are of mammalian origin. Non-limiting examples of suitable mammalian species include mice, rats, rabbits, hamsters, gerbils, guinea pigs, dogs, cats, goats, pigs, cows, horses, donkeys, sheep, camels, llamas, dolphins, whales, prosimians, monkeys, lesser apes, and great apes. In preferred embodiments, the immunoinhibitory complex bound by the allosteric modulator of the present invention comprises one or more subunits derived from a human (Homo sapiens). In further embodiments, said subunits include naturally occurring isoforms, polymorphic variants, mutants, or engineered derivatives thereof, as discussed in detail below. In some embodiments, the allosteric modulator disclosed herein may target hybrid or chimeric protein complexes composed of subunits from different species (e.g., human-mouse chimeras), or may exhibit cross-species reactivity.

It shall be understood that the allosteric modulators disclosed herein may target complexes composed of naturally occurring proteins and/or non-naturally occurring proteins, provided that such proteins are capable of forming immunoinhibitory complexes, as defined herewith. As used herein, the term "non-naturally occurring proteins" refers to proteins that have been synthetically produced, artificially designed, genetically modified and/or otherwise engineered or altered by human intervention. These include, without limitation, recombinant proteins, chimeric constructs, fusion proteins, mutant variants, chemically modified polypeptides, and de novo designed sequences. In certain embodiments, the non-naturally occurring protein comprises an amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to a naturally occurring (wild-type) sequence of a known inhibitory surface immune receptor, ligand, or accessory molecule, as determined by standard sequence alignment algorithms (e.g., BLAST, ClustalW, or equivalent). In various embodiments, such proteins may incorporate one or more modifications relative to the wild-type sequence, including but not limited to amino acid substitutions, deletions (e.g., N-terminal, C-terminal, or in internal regions such as loops or linker segments), insertions, duplications, or additions of non-native sequences. A modified protein comprised within an immunoinhibitory complex targeted by the allosteric modulator of the present invention may also be presented as part of a conjugate molecule, such as a fusion with another protein domain, peptide, tag, small molecule, or polymer. Combinations of such modifications are also included. Additionally, the immunoinhibitory complexes targeted by the allosteric modulators of this invention may comprise proteins that represent natural variants, including but not limited to polymorphic forms, splice variants (resulting from alternative splicing events), allelic variants, post-translationally modified isoforms, or interspecies homologues (e.g., orthologs and paralogs from model organisms or closely related species). For example, the PD-1 »PD-L1 complex may comprise a membrane-bound PD-L1 isoform or a soluble PD-L1 isoform¹⁴⁰. In certain embodiments, the immunoinhibitory complex may include functionally active mutants, gain- or loss-of-function variants, or rare alleles associated with pathological or protective phenotypes. Accordingly, the invention encompasses allosteric modulators that target/bind to immunoinhibitory protein complexes of broad structural and functional diversity, whether composed of wild-type proteins or engineered variants, provided that such complexes are capable of presenting a modulator-specific epitope suitable for selective allosteric binding, as described herewith.

In various embodiments, the allosteric modulator disclosed herein selectively binds to a protein complex comprising at least one inhibitory immune receptor that is localized on the surface of an immune cell. The immune cell may be either a mature (fully differentiated) or immature (not fully differentiated) cell of the immune system. In certain embodiments, the immune cell is of lymphoid lineage. Non-limiting examples of such cells include: (i) T lymphocytes (T cells), including but not limited to helperT cells (Th), cytotoxic T cells (Tc), and regulatoryT cells (Tregs); (ii) B lymphocytes (B cells); (iii) natural killer (NK) cells; (iv) innate lymphoid cells (ILCs); (v) y6 T cells (gamma delta T cells); (vi) natural killer T (NKT) cells; and (vii) innate-like T cells, including mucosal-associated invariantT (MAIT) cells and invariant NKT (iNKT) cells. In other embodiments, the immune cell is of myeloid lineage. Non-limiting examples of such cells include macrophages, dendritic cells, neutrophils, monocytes, mast cells, eosinophils, and basophils.

In another embodiment, the allosteric modulator of the present invention specifically binds to an immunoinhibitory complex comprising at least one inhibitory surface immune receptor selected from those listed in Figure 1, and at least one corresponding ligand selected from those listed in Figure 2. As used herein, the term "corresponding ligand" refers to a ligand known to physiologically and/orexperimentally interactwith a given receptorto form an immunoinhibitory complex. Suitable inhibitory immune receptors include, but are not limited to, PD-1 , BTLA, BTN2A2, CD112R, CD160, CD200R, CD244, CD32B, CD33, CD5, CD79a, CD96, CEACAM-1 , CTLA-4, FCRL4, FDF03, KIR2DL1 , KIR2DL2, KIR2DL3, KIR2DL5, KIR3DL1 , KIR3DL2, KLRB1 , KLRG1 , LAG-3, LAIR-1 , LILRB1 , LILRB2, LILRB4, LILRB5, NCTR2, NKG2A, N rp1 , PECAM-1 , Plexin-A4, Sema3A, SIRPa, SLAMF6, TIGIT, TIM- 3, TNFR2, VISTA, VSIG-4, and VSTM1. Suitable ligands of inhibitory immune receptors include, without limitation, PD-L1 , PD-L2, adiponectin, Angptl2, APLP2, B7-H4, BTN1A1 , C1Q, C3b, cadherin 4, CD112, CD155, CD1 d, CD200L, CD324, CD325, CD38, CD47, CD48, CD5, CD72, CD79b, CD80, CD86, CD99, CEACAM-1 , CEACAM8, CLC2D, CLEC4G, collagen I, collagen III, Collectin-12, EFNB1 , Gal-1 , Gal-8, Gal-9, HLA-A24, HLA-B27, HLA-B27 FHC, HLA-B57, HLA-B7 FHC, HLA-C, HLA-C2, HLA-Cw1 , HLA-Cw3, HLA-Cw7, HLA-E, HLA-G, HVEM, IgA, IgG, IGSF21 , ITGAV, ITGB3, IYD, KIRREL3, MHC II, mTNFa, NPDC1 , PCNA, PECAM-1 , PIANP, PSGL-1 , PVR, PVRL2, Sema4A, SLAMF6, SMPD1 , SP-D, TIM-3, and VSIG-3.

In an alternative embodiment, the allosteric modulator of this invention specifically binds to a complex set forth in Figure 3. Examples of suitable complexes include, without limitation, PD- 1 *PD-L1 , PD-1 ’PD-L2, BTLA’HVEM, CD200R*CD200L, TIGIT*CD112, TIGIT’PVR, TNFR2*mTNFa, PD-1 *CD48, LAIR-1 *C1 Q, LAIR-1 ’SP-D, LAIR-1 ’adiponectin, LAIR-1 ’collagen-l, LAIR-1 ’collagen- III, LAIR-1 ’EFNB1 , CD33’C1q, PD-1 ’BTN1A1 , TIM-3’Gal-9, CTLA-4’CD80, NCTR2’PCNA, CTLA- 4»CD86, CD160’HVEM, VSIG-4’C3b, VISTA’VSIG-3, VISTA’PSGL-1 , LAG-3*MHC-II, CD112R’PVRL2, VISTA’Gal-9, KIR2DL2*HLA-C, KIR2DL3’HLA-Cw1 , KIR2DL3’HLA-Cw3, KIR2DL3’HLA-Cw7, KIR3DL2*HLA-B27, KIR2DL1 ’HLA-C2, KIR3DL1 ’HLA-B57, KIR3DL1 ’HLA-B27, KIR3DL1 ’HLA-A24, KIR2DL5’CD155, VSTM1 ’Gal-1, SIRPa*CD47, LILRB2*HLA-G, LILRB2*CD1d, LILRB2’Angptl2, LILRB1 ’HLA-G, LILRB4’Gal-8, LILRB5’HLA-B27 FHC, LILRB5’HLA-B7 FHC, Nrp1 ’B7-H4, Plexin-A4’B7-H4, Sema3A’B7-H4, Nrp1 ’Sema4A, CD5’CD5, CD5’CD72, KLRB1 ’SMPD1 , KLRB1 ’CLC2D, KLRG1 ’CD324, KLRG1 ’CD325, KLRG1 ’Cadherin 4, PECAM-1 ’ ITGAV’ITGB3, PECAM-1 *CD38, PECAM-1 ’PECAM-1 , CEACAM-1 ’CEACAM-1 , CEACAM-1 ’TIM-3, CD32B*lgG, FDF03*PIANP, FDF03*Collectin-12, FDF03*CD99, FDF03’NPDC1 , FDF03*CLEC4G, CD96*CD155, NKG2A*HLA-E, CD244*CD48, CD244’IYD, CD244’IGSF21 , FCRL4*lgA, SLAMF6*SLAMF6, CD79a*CD79b, LILRB5*APLP2, CD33*KIRREL3, SIRPa*CD99, and BTN2A2*CEACAM8.

Thus, in various embodiments, the allosteric modulator of the present invention specifically binds to an immu noin hibitory complex comprising at least one inhibitory surface immune receptor and at least one ligand. In this context, an inhibitory immune receptor is an immune receptor that, upon binding to its designated orthosteric ligand, transmits a signal that downregulates or suppresses the immune response. Inhibitory immune receptors play a crucial role in maintaining immune homeostasis, preventing immune hyperreactivity, and protecting against autoimmunity¹⁴¹. They may either function independently or serve as regulators of the signal strength of immunostimulatory receptors. In this context, a subset of inhibitory receptors encompasses immune checkpoint receptors, which act as inhibitory co-receptors for the TCR or BCR. With few exceptions, inhibitory receptors transmit their repressive signals through one or more i) immunoreceptor tyrosine-based inhibitory motifs (ITIMs), and/or ii) immunoreceptor tyrosinebased switch motifs (ITSMs) located in the cytoplasmic domains. These recruit downstream phosphatases, which attenuate or abrogate concomitant activation signals⁹⁶. For example, the cytoplasmic tail of PD-1 contains two tyrosine-based structural motifs: an ITIM and an ITSM. PD-1 signaling begins when these motifs are phosphorylated, for instance upon co-engagement of PD-1 and TCR with their respective ligands. This triggers the recruitment and activation of Src homology region 2 domain-containing phosphatases (SHPs), which dampen the phosphorylation-dependent signaling cascades downstream of the TCR and the co-stimulatory receptor CD28. As a result, this suppresses cellular T cell functions such as activation, proliferation, cytotoxicity, and cytokine production¹⁴². Notably, while some inhibitory/co-inhibitory receptors exclusively inhibit specific activating signals, others modulate multiple immunostimulatory pathways. For instance, PD-1 counteracts TCR signaling as well as CD28 signaling¹⁴³.

In one embodiment, the allosteric modulator of this invention binds to the hPD-1 »hPD-L1 complex. PD-1 (Programmed cell death-1 ) also known as CD279, is a type I transmembrane protein that belongs to the CD28 immunoglobulin superfamily. It serves as a critical mediator of immune tolerance within both the central and peripheral immune systems. PD-1 is predominantly expressed on various immune cells, including T cells, B cells, macrophages, dendritic cells, and natural killer (NK) cells. Encoded by the PDCD1 gene, PD-1 consists of three main domains: an extracellular immunoglobulin variable (IgV)-like domain, a transmembrane domain, and a cytoplasmic tail. The extracellular domain may bind two ligands - PD-L1 (also known as B7-H1 or CD274) and PD-L2 (CD273) - which are typically found on antigen-presenting cells and tumor cells. The cytoplasmic region of PD-1 contains two key signaling motifs: the immunoreceptor tyrosinebased inhibitory motif (ITIM) and the immunoreceptor tyrosine-based switch motif (ITSM). Upon ligand engagement, these motifs become phosphorylated by kinases such as Lek, Src, or Lyn, leadingtothe recruitment of SHP-2, a tyrosine phosphatase. SHP-2 dephosphorylates downstream signaling molecules such as ZAP70, PI3K, and Ras, thereby attenuating signaling through the T cell receptor (TCR) and the co-stimulatory molecule CD28.

The PD-1/PD-L1 axis plays a fundamental role in maintaining immune homeostasis. It acts as a checkpoint to prevent excessive immune activation, which could otherwise lead to tissue damage or autoimmunity. Disruption of this pathway has been linked to hyperactive immune responses and the development of autoimmune diseases. PD-1 expression is tightly regulated on T cells. Under resting conditions, naive T cells express minimal levels of PD-1. However, during acute immune responses, its expression is rapidly upregulated to help balance immune activation and tissue protection. For instance, PD-1 -deficient mice clearviral infections more rapidly but also sufferfrom increased tissue damage. In chronic infections, sustained PD-1 expression is associated with T cell exhaustion, characterized by reduced cytokine production, impaired proliferation, and weakened cytotoxic responses. Tumor cells often exploit this immune checkpoint pathway to avoid immune detection. By overexpressing PD-L1 or PD-L2, tumor cells engage PD-1 on infiltrating? cells, leading to T cell dysfunction and immune evasion.

The allosteric modulator of the present invention, which specifically binds to an epitope unique for and/or predominantly present on a protein complex comprising at least one inhibitory surface immune receptor and its ligand, induces positive cooperativity within said complex upon binding. Positive cooperativity refers to a phenomenon wherein binding of the allosteric modulator enhances the affinity and/or binding kinetics of one or more molecular partners within the protein complex - such as the immune receptor, its ligand, and/ or additional modulator molecules - thereby stabilizing the complex’s functional state, as described in detail herein.

The positive cooperativity induced by the allosteric modulator of the present invention is characterized by a cooperativity factor a (alpha) having a value greater than 1 (a > 1 ), as described herein. The cooperativity factor a describes heterotropic allosteric interactions, reflecting how the binding of one ligand type - such as the allosteric modulator of this invention - affects the binding affinity or kinetics of a different ligand type within a protein complex (e.g., of a ligand of an inhibitory surface immune receptor). An a value greater than 1 indicates that binding of the allosteric modulator enhances the binding of the distinct ligand, thereby demonstrating positive allosteric modulation of the immune complex.

Byway of example, the allosteric modulator of the present invention that specifically binds to the PD-1 »PD-L1 complex enhances the binding affinity of PD-L1 to PD-1 within said complex. This enhancement is quantitatively demonstrated by a cooperativity factor a greater than 1 , measured for the PD-1 »PD-L1 interaction in the presence of the allosteric modulator. In such embodiments, a ternary complex is formed comprising the immune receptor (PD-1 ), its ligand (PD-L1), and the allosteric modulator (i.e. , PD-1 »PD-L1 •modulator). Underthese circumstances, the cooperativity factor a is defined as the ratio of the equilibrium dissociation constant (K_D) for the binary receptorligand complex (PD-1 »PD-L1) to the equilibrium dissociation constant (K_D) for the ternary complex (PD-1 »PD-L1 •modulator). An a value greater than 1 indicates that the presence of the allosteric modulator increases the affinity between PD-1 and PD-L1 , thereby demonstrating positive heterotropic cooperativity, as defined herein. It shall be understood that these principles equally apply to other binary immunoinhibitory receptor-ligand complexes targeted by the allosteric modulators of this invention. That is, for any such binary receptor-ligand complex, binding of the allosteric modulator can enhance the interaction affinity within the complex, as reflected by a cooperativity factor a greater than 1 .

In further embodiments, positive cooperativity is additionally defined by a Hill coefficient (n_H) having a value greater than 1 . As defined herein, the Hill coefficient (n_H) is a parameter commonly employed to describe cooperative binding behavior in multimeric protein complexes exhibiting homotropic interactions, wherein multiple binding sites for the same ligand exist within a complex/molecule. A Hill coefficient (n_H) greater than 1 indicates that the binding of one ligand molecule to a site increases the likelihood or affinity of subsequent binding events by identical ligand molecules to other equivalent sites within the complex, thereby generating a sigmoidal binding curve characteristic of positive cooperativity¹⁴⁴.

By way of a non-limiting example, the binding of B7.1 to the CTLA-4 dimer may be enhanced by the allosteric modulator of the present invention. Specifically, the binding of the first B7.1 molecule to one CTLA-4 monomer within the CTLA-4* CTLA-4 dimer can be positively influenced by the allosteric modulator, as measured by a cooperativity factor a greater than 1. Subsequently, the binding of a second B7.1 molecule to the other CTLA-4 monomer within the CTLA-4* CTLA-4 dimer may be further enhanced by the first B7.1 binding event, demonstrating positive cooperativity characterized by a Hill coefficient (n_H) greater than 1. Together, these parameters provide complementary measures of cooperative phenomena within immunoinhibitory receptor-ligand complexes: the Hill coefficient (n_H) measures cooperative binding of identical ligands to multiple sites ("homotropic cooperativity", as defined herewith), while the cooperativity factor a quantifies allosteric interactions between distinct ligand types ("heterotropic cooperativity", as defined herewith). Both metrics are valuable for evaluating the functional modulation of immunoinhibitory complexes by the allosteric modulators disclosed herein.

It shall be further understood that, in some embodiments, binding of the allosteric modulator disclosed herein to a multimeric protein complex- such as the CTLA-4*CTLA-4*B7-1 *B7-1 complex - may also enhance the binding of additional identical allosteric modulator molecules to other binding sites within the same complex. Such an effect is encompassed within the scope of positive cooperativity and is characterized by a Hill coefficient value greater than 1 , reflecting cooperative bindingof identical allosteric modulator molecules to multiple sites within the multimeric complex.

In one embodiment, the invention provides an allosteric modulator that enhances immunosuppressive signaling of an immunoinhibitory complex, for example the hPD-1 *hPD-L1 complex. Specifically, the allosteric modulator disclosed herein binds selectively to an epitope present on said complex, and, through the induction of positive cooperativity, as defined herein, promotes or reinforces a conformational state that favors sustained engagement between the complex constituents. This conformational shift facilitates or stabilizes the active signaling conformation of the inhibitory receptor in complex with its endogenous ligand, thereby enhancing, or inducing, or promoting the activity of downstream inhibitory signaling cascades.

In various embodiments, the modulation of downstream signaling by the allosteric modulator disclosed herein is achieved through enhanced affinity between the inhibitory receptor (such as hPD-1 ) and its ligand (such as hPD-L1), as measured by factor alpha (a) greater than 1 , as described herewith in detail. Additionally or alternatively, said modulation of downstream signaling may be achieved through improved efficacy of immunoinhibitory complex, as measured by factor 6 (delta) greater than 1 . Factor 6 reflects the influence of the allosteric modulator-ligand interaction on the functional activation state of the receptor, independent of the effects on the binding affinity. In this context, 6 quantifies how binding of the modulator impacts the receptor’s transition to an active signaling conformation upon ligand engagement, thus providing a complementary and functionally meaningful metric of cooperative allosteric modulation by the modulator of this invention. Thus, the allosteric modulator disclosed herein may function as an efficacy modulator, capable of inducing a conformational change within a receptor engaged in the target complex (for example, hPD-1 »hPD-L1 ) that is transmitted to intracellular domains involved in signal transduction, thereby modulating the receptor’s downstream signaling capacity.

In various embodiments, the allosteric modulator of this disclosure functions as a positive allosteric modulator (PAM) that enhances the effects of a ligand bound to the orthosteric site of an immunoinhibitory complex, such as hPD-1 »hPD-L1 . Such PAMs exhibit positive cooperativity with the orthosteric ligand, thereby increasing the receptor’s affinity for the ligand (factor alpha), enhancing the potency of downstream signaling events. Such PAMs may also improve receptor efficacy, which refers to the magnitude of the response triggered by activation through the orthosteric ligand (factor delta). As such, the PAM of this invention stabilizes immunoinhibitory ligand-receptor complexation and promotes immunosuppressive signaling.

In further embodiments, the allosteric modulator of this invention exhibits spatiotemporal selectivity for the immunoinhibitory complex. By "spatiotemporal selectivity" is meant that the modulator acts specifically on naturally formed receptor-ligand complexes, thereby rendering its activity dependent on the presence of the ligand and the formation of the immunoinhibitory complex in situ. Consequently, the modulator’s functional effects are restricted to the locations and timeframes in which the immunoinhibitory interaction naturally occurs. This spatiotemporal selectivity ensures that the allosteric modulator of this invention exerts its effects only in tissues and/or microenvironments where the receptor and ligand interact physiologically or pathologically. Non-limiting examples of such tissue environments include inflamed tissues where immune responses are active, primary and secondary lymphoid organs such as the thymus, bone marrow, lymph nodes, and spleen, as well as tertiary lymphoid structures that can form in peripheral tissues during chronic inflammation and/or autoimmune responses. Thus, the modulator’s spatiotemporal selectivity provides a targeted mechanism of action, minimizing off-target effects and systemic immune modulation by confining its activity to relevant immune microenvironments where the immunoinhibitory complexes are present and engaged.

In some embodiments, the allosteric modulator of this disclosure binds to immunoinhibitory complexes formed specifically at the immune synapse. The immune synapse is a specialized, dynamic interface between immune cells where receptor-ligand interactions are spatially and temporally organized to regulate immune activation or inhibition¹⁴⁵. Typical immune synapses involve, for example, T cells bearing inhibitory immune receptors such as PD-1 , CTLA-4, or BTLA, interacting with antigen-presenting cells (APCs) - including dendritic cells, macrophages, and B cells - that express corresponding ligands such as PD-L1 , B7-1 , B7-2, or HVEM. Beyond classical T cell-APC synapses, immune synapses can also form between natural killer (NK) cells and their target cells¹⁴⁶. Similarly, B cells can form synapse-like contacts with follicular dendritic cells or T follicular helper cells, where regulatory receptor-ligand complexes modulate B cell activation and antibody responses ¹⁴⁷.

By enhancing the cooperativity of immunoinhibitory complexes, the allosteric modulators disclosed in this application modulate downstream signalling to promote immunosuppressive pathways. As such, they hold strong therapeutic potential for conditions characterized by excessive or dysregulated inflammatory responses, including autoimmune diseases. Importantly, these modulators preserve the native spatial and temporal dynamics of immune signaling, supporting receptor activation mediated by endogenously formed ligand-receptor interactions. In contrast to traditional receptor agonists that elicit broad, systemic stimulation of immune pathways, the modulators described herein selectively enhance natural, localized immune responses - specifically at sites of inflammation or at key immunological interfaces such as those between T cells and antigen-presenting cells (APCs) in secondary or tertiary lymphoid tissues. This precision and context-dependent mechanism of action confer significant therapeutic advantages, offering a more physiologically aligned and potentially safer alternative to existing treatment modalities, as further discussed in the Background section. In various embodiments, the allosteric modulator described herein specifically binds to a conformational epitope present on the target immunoinhibitory complex. Depending on the oligomeric state of the complex, the modulator’s binding specificity and mode of interaction may vary. For dimeric complexes, such as the PD-1 »PD-L1 complex, the allosteric modulator may recognize and bind to conformational epitopes located on: i) both subunits of the complex, meaning both the receptor and the ligand molecules involved in the complex, or ii) a single subunit of the complex, i.e., either the receptor molecule or the ligand molecule of the complex. In the case of trimeric complexes, such as the BTLA»HVEM»CD160 complex, the allosteric modulator may exhibit a broader range of binding modalities. That is, the modulator may bind to conformational epitopes located on: i) all three subunits of the complex, ii) any two of the subunits forming the complex, oriii) a single subunitwithin thetrimeric complex. Forquaternary complexes, exemplified by the CTLA-4»CTLA-4»B7-1 »B7-1 complex, the allosteric modulator may recognize and bind conformational epitopes located on: i) all four subunits of the complex, ii) any three subunits of the complex, iii) any two subunits of the complex, or iv) a single subunit within the quaternary assembly. Therefore, in various embodiments, the allosteric modulator may bind to any number of immune complex-associated epitopes, which may be located on: i) all subunits of the complex, ii) a subset of subunits within the complex, or iii) a single subunit of the complex. It shall be understood that the induction of positive cooperativity within immune complexes by the allosteric modulators of this invention can be achieved irrespective of the number or combination of conformational epitopes recognized and bound by the modulator.

In a further specific embodiment described herein, the allosteric modulator specifically binding the immunoinhibitory surface receptor-ligand complex hPD-1 -hPD-L1 comprises an ISVD that binds to an epitope located on hPD-1 , comprising at least one or more of the residues V43, A72, L73, V75, E141 , and/or T177 of hPD-1 protein as provided in SEQ ID NO: 08. More preferably, said epitope, also provided herein as epitope B, comprises at least residues V43 and E141. In another embodiment described herein, the allosteric modulator specifically binding the immunoinhibitory surface receptor-ligand complex hPD1 -hPDL1 comprises an ISVD that binds to a junctional or connecting epitope with residues of hPD-1 and hPD-L1 being involved in the interaction with the ISVD, and comprising at least the residue E61 and/or S62 of hPD-1 as provided in SEQ ID NO: 08, and/or as provided herein as epitope A.

In a further aspect of the invention, the allosteric modulator of this invention specifically binds to the hPD-1 »hPD-L1 complex through at least one or more, preferably 3 complementaritydetermining region(s) (CDR(s)), wherein the CDR comprises any of the amino acid sequences as depicted in SEQ ID NOs: 76, 77, or 78. In one embodiment, said CDR1 comprises or is provided as a sequence shown in SEQ ID NO: 76, wherein X1 = R, or P; and X2 = T, or G; and X3 = S, or N; and X4 = Y, D, A, S, R, orT; and X5 = Y, V, orT; and/or CDR2 comprises or is provided as a sequence shown in SEQ ID NO: 77, wherein Y1 = I, G, V, or S; and Y2 = T or A; and/or CDR3 comprises or is provided as a sequence shown in SEQ ID NO: 78, wherein Z1 = P or A; and Z2 = S, A, or F; and Z3 = A, E, D, T, E, or G.

In further embodiments, the allosteric modulator of this disclosure comprises an ISVD that comprises the complementarity-determining regions (CDRs) as present in SEQ ID NOs: 09, 10, 11 , 12, 13, 14, 15, 32, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, or 48, wherein the CDRs are annotated according to the Kabat, MacCallum, IMGT, AbM, or Chothia CDR annotation system.

In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises or corresponds to a sequence wherein CDR1 comprises or is SEQ ID NO: 17, CDR2 comprises or is SEQ ID NO: 18, CDR3 comprises or is SEQ ID NO: 19. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 20, CDR2 comprises or is SEQ ID NO: 21 , CDR3 comprises or is SEQ ID NO: 22. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 79, CDR2 comprises or is SEQ ID NO: 80, CDR3 comprises or is SEQ ID NO: 81. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 82, CDR2 comprises or is SEQ ID NO: 83, CDR3 comprises or is SEQ ID NO: 84. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 85, CDR2 comprises or is SEQ ID NO: 86, CDR3 comprises or is SEQ ID NO: 87. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 88, CDR2 comprises or is SEQ ID NO: 89, CDR3 comprises or is SEQ ID NO: 90. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 91 , CDR2 comprises or is SEQ ID NO: 92, CDR3 comprises or is SEQ ID NO: 93. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 94, CDR2 comprises SEQ ID NO: 95, CDR3 comprises or is SEQ ID NO: 96. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 97, CDR2 comprises or is SEQ ID NO: 98, CDR3 comprises or is SEQ ID NO: 99. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 100, CDR2 comprises or is SEQ ID NO: 101 , CDR3 comprises or is SEQ ID NO: 102. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 103, CDR2 comprises or is SEQ ID NO: 104, CDR3 comprises or is SEQ ID NO: 105. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 106, CDR2 comprises or is SEQ ID NO: 107, CDR3 comprises or is SEQ ID NO: 108. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 106, CDR2 comprises or is SEQ ID NO: 107, CDR3 comprises or is SEQ ID NO: 109. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 106, CDR2 comprises or is SEQ ID NO: 104, CDR3 comprises or is SEQ ID NO: 110. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 106, CDR2 comprises or is SEQ ID NO: 111 , CDR3 comprises or is SEQ ID NO: 112. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 106, CDR2 comprises or is SEQ ID NO: 113, CDR3 comprises or is SEQ ID NO: 108. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 114, CDR2 comprises or is SEQ ID NO: 115, CDR3 comprises or is SEQ ID NO: 116. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 106, CDR2 comprises or is SEQ ID NO: 111 , CDR3 comprises or is SEQ ID NO: 117. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 106, CDR2 comprises or is SEQ ID NO: 111 , CDR3 comprises or is SEQ ID NO: 118. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 119, CDR2 comprises or is SEQ ID NO: 111 , CDR3 comprises or is SEQ ID NO: 112. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 120, CDR2 comprises or is SEQ ID NO: 111 , CDR3 comprises or is SEQ ID NO: 112. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 106, CDR2 comprises or is SEQ ID NO: 107, CDR3 comprises or is SEQ ID NO: 121. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 122, CDR2 comprises or is SEQ ID NO: 111 , CDR3 comprises or is SEQ ID NO: 123. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 124, CDR2 comprises or is SEQ ID NO: 111 , CDR3 comprises or is SEQ ID NO: 109. In another embodiment, the allosteric modulator of this disclosure comprises an ISVD, and said ISVD comprises a sequence wherein CDR1 comprises or is SEQ ID NO: 125, CDR2 comprises or is SEQ ID NO: 126, CDR3 comprises or is SEQ ID NO: 127.

In further embodiments, the allosteric modulator of this disclosure comprises an ISVD wherein the ISVD comprises an amino acid sequence selected from any one of SEQ ID NOs: 09, 10, 11 , 12, 13, 14, 15, 32, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, or 48.

In alternative embodiments, the allosteric modulator of this disclosure comprises an ISVD, wherein the ISVD comprises an amino acid sequence having at least 90% sequence identity over the full length of the ISVD as present in any of SEQ ID NO: 09, 10, 11 , 12, 13, 14, 15, 32, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, or 48. In such embodiments, said "amino acid sequence having at least 90% sequence identity over the full length of the ISVD" is a sequence of a fully functional variant of the allosteric modulator of this invention. Said functional variant retains the antigenbinding specificity for hPD-1 »hPD-L1 , and may include conservative amino acid substitutions that do not substantially impair the binding affinity or structural integrity.

In other embodiments, the allosteric modulator of this disclosure comprises an ISVD wherein the ISVD comprises an amino acid sequence that has been subject to humanization and/or optimization, as described herewith. For example, said humanization and/or optimization may be achieved by substitution of framework residues with corresponding human germline residues, such as from the IGHV3 family, to reduce immunogenicity while maintaining or enhancing biophysical properties, such as solubility, stability, or expression yield, and/or functional properties such as target binding affinity or virus neutralization potency. In this context, the humanized and/or functional variants of the ISVD-comprising modulator of this invention are obtained as described herein, and may be based on primary sequence alignment with the human IGHV3 coding sequence, wherein one or more key residues within the alpaca-derived framework regions of the VHHs are substituted with corresponding residues from the human germline sequence. This substitution is followed by biophysical and functional analyses of the resultingVHH variants after recombinant expression. Specifically, the stability, solubility, and neutralizing capacity of the modified VHHs are evaluated using standard in vitro and/or in vivo assays. The humanized variants retain the antigen-binding specificity and affinity of the original VHH, while exhibiting improved compatibility with the human immune system, reduced immunogenicity, and favorable pharmacokinetic properties.

In various embodiments, the allosteric modulator of this disclosure comprises an ISVD wherein the ISVD comprises an amino acid sequence that has been subject to humanization and said sequence may be selected from any of the following SEQ ID NOs: 49, 50, 51 , 53, 54, 55, 56, 57, 58, or 128-184. Moreover, it is clear from the disclosure that the exemplified humanized variants allow for framework residue substitutions as typically known in the art. So in a further embodiment, the humanized variants provided herein are selected from the ISVD comprising the CDRs according to the original camelid VHH sequence, with framework region sequences that allow for substitutions in the original FR based on the working embodiments shown herein, or basically any combination of working substituted residue in any possible ways. For instance, when in hypothetical humanized variant 1 FR1 has a substitution of 5V, and in hypothetical humanized variant 2 FR1 has a substitution of 11 P, these combination of both substitutions is disclosed herein as a further alternative humanization. Further, if hypothetical humanized variant 1 FR1 has a substitution of 5V and 1 D, and hypothetical humanized variant 2 FR1 has a substitution of 1 E and 11 P, the further humanized variant combining 1 E and 5V is also disclosed herein.

In further embodiments, the allosteric modulator of this disclosure comprises an ISVD wherein the ISVD comprises an amino acid sequence selected from anyone of SEQ ID NOs: 245-248, 253-262, or 263-266.

In various embodiments, the allosteric modulators of this disclosure are positive allosteric modulators of immunoinhibitory complexes, such as hPD-1 »hPD-L1 or hTIGIT»hPVR, without limitation. For instance, T cell immunoreceptor with Ig and ITIM domains (TIGIT) is a transmembrane protein expressed on multiple immune cell types, including CD8+ and CD4+ T cells, regulatory ? cells (Tregs), and natural killer (NK) cells. TIGIT is an inhibitory receptor whose ligand, PVR (CD155), is expressed on parenchymal cells as well as antigen-presenting cells (APCs)¹⁴⁸. TIGIT signaling functions to suppress T-cell activation and proliferation, thereby promoting immune tolerance, particularly in the context of autoimmune diseases¹⁴⁹. TIGIT is a critical immune checkpoint that plays a key role in maintaining immune tolerance and preventing autoimmunity^{12 150}. For example, studies have shown that TIGIT expression in CD4⁺ T cells is reduced in the synovial fluid of patients with active rheumatoid arthritis (RA), and that overexpression of TIGIT suppresses the production of IFN-y and IL-17 by RA CD4⁺ T cells¹⁵¹. TIGIT expression is impaired in CD4⁺ T cells from patients with autoimmune polymyositis (PM)¹⁵². Consistently, cell-intrinsic TIGIT deficiency in these patients leads to exaggerated Th1 and Th17 responses, implicatingTIGIT dysfunction in the pathogenesis and progression of myositis. A study by Kojima M et al.¹⁵³ investigated the effects of an agonistic anti-human TIGIT monoclonal antibody in several autoimmune disease models. The mAb suppressed the activation of CD4⁺ T cells, particularly follicular helper (Tfh) and peripheral helper (Tph) cells that expressed high levels of TIGIT. It also enhanced the suppressive function of naive regulatory T cells, suggesting a dual mechanism of action. These findings indicate that enhancing TIGIT signaling restores T cell balance and alleviates autoimmune symptoms, highlighting its therapeutic potential in those indications.

According to a preferred embodiment, the allosteric modulators of this invention are derived from the innate or adaptive immune system. Specifically, these binding agents are preferably derived from immunoglobulins, with antibodies or antibody fragments being particularly preferred. The term "antibody" (Ab), as defined herein, broadly refers to a polypeptide encoded by an immunoglobulin gene or a functional fragment thereof that specifically binds and recognizes an antigen, as understood by those skilled in the art. An antibody encompasses conventional four- chain immunoglobulins composed of two identical pairs of polypeptide chains, each pair consisting of one "light" chain (of approximately 25 kDa) and one "heavy" chain (of approximately 50 kDa). Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen-binding site.

The term “antibody” also encompasses whole antibodies, including single-chain whole antibodies, as well as antigen-binding fragments. Antigen-binding fragments may include, but are not limited to, Fab, Fab', and F(ab')2 fragments, Fd fragments, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (dsFv), fragments comprising or consisting of either a VL orVH domain, and any combination thereof or any other functional portion of an immunoglobulin peptide capable of binding to the target antigen. The term "antibody" also encompasses heavy chain antibodies or fragments thereof, including immunoglobulin single variable domains, as further defined in this application.

According to a particularly preferred embodiment, the binding agent of this disclosure comprises an immunoglobulin single variable domain, specifically a Nanobody™ (Nb, nanobody). Nanobodies include, but are not limited to, VHH domains derived from camelid heavy chain antibodies. The term Nanobody, as defined herein, refers to a single-domain antigen-binding fragment naturally occurring in heavy chain-only antibodies found in camelids. Such single variable domains are well known and widely utilized in the art.

In alternative embodiments, the allosteric modulator of this disclosure comprises an ISVD wherein the ISVD comprises an amino acid sequence that has been subject to humanization and/or optimization, as described herewith. For example, said humanization and/or optimization may be achieved by substitution of framework residues with corresponding human germline residues, such as from the IGHV3 family, to reduce immunogenicity while maintaining or enhancing biophysical properties, such as solubility, stability, or expression yield, and/or functional properties such as target binding affinity or virus neutralization potency. In this context, the humanized and/or functional variants of the ISVD-comprising modulator of this invention are obtained as described herein, and may be based on primary sequence alignment with the human IGHV3 coding sequence, wherein one or more key residues within the alpaca-derived framework regions of the VHHs are substituted with corresponding residues from the human germline sequence. This substitution is followed by biophysical and functional analyses of the resultingVHH variants after recombinant expression. Specifically, the stability, solubility, and neutralizing capacity of the modified VHHs are evaluated using standard in vitro and/or in vivo assays. The humanized variants retain the antigen-binding specificity and affinity of the original VHH, while exhibiting improved compatibility with the human immune system, reduced immunogenicity, and favorable pharmacokinetic properties.

In another embodiment, any of the allosteric modulators of an immunoinhibitory complex, as described herein, may be further labeled, tagged, or fused to an additional moiety, such as a detection moiety, a functional or therapeutic moiety. The conjugated functional moiety may include, but is not limited to, a diagnostic label, an imagingagent, a cytotoxic agent, a radioisotope, an enzyme, a drug molecule, or a pharmacologically active compound. Alternatively, or in addition, the conjugated moiety may serve a pharmacokinetic or pharmacodynamic purpose, such as enhancing serum stability, tissue penetration, or systemic half-life. Non-limiting examples of such half-life extension moieties include albumin-binding domains, serum albumin, polyethylene glycol (PEG) or derivatives thereof (such as methoxypoly(ethyleneglycol) or mPEG), XTEN polypeptides, Fc domains, or fusion to other long-lived plasma proteins.

Another technique for increasing the half-life of a binding agent may comprise the engineering into bifunctional or bispecific domains (for example, one or more ISVDs or active antibody fragments against the protein complex coupled to one ISVD or active antibody fragment against serum albumin or pulmonary surfactant protein A (Spa) aiding in prolonging half-life)) or into fusions of antibody fragments, in particular immunoglobulin single variable domains, with peptides (for example, a peptide against a serum protein such as albumin). The coupling to additional moieties will result in multispecific binding agent, as further disclosed herein. The conjugation or fusion may be achieved by covalent linkage, chemical cross-linking, recombinant fusion, or other suitable methods known in the art, and is preferably selected so as not to impair the binding specificity or biological activity of the ISVD described herewith.

In a particular further embodiment, the allosteric modulator of the invention is provided in a “multivalent” or “multispecific” format. These formats are generated by bonding two or more monovalent ISVDs, which may be identical or non-identical, via chemical conjugation or recombinant DNA techniques. The individual ISVDs may be directly fused, connected via flexible or rigid linker sequences, or joined through fusion with Fc domain-encoding sequences, including wild-type, engineered, or extended half-life Fc variants. Non-limiting examples of such multivalent constructs include: bivalent constructs (e.g., ISVD1-linker-ISVD1 or ISVD1-Fc-ISVD1), trivalent constructs (e.g., tandem repeats or ISVD1-ISVD2-ISVD1), tetravalent constructs, and higher-order multimeric assemblies. Such multivalent constructs can enhance avidity, functional valency, target clustering, and signal modulation, and may be tailored for increased potency, selectivity, or pharmacokinetic properties. The ISVDs within these constructs may recognize distinct epitopes, and/or different conformational states.

Moreover, the ISVD-comprising modulator of this invention, or any of its humanized or optimized variants, may be genetically or chemically fused - either directly or via a suitable linker - to generate bivalent or multivalent constructs. Such constructs may include tandem repeats or head-to-tail fusions, terms used interchangeably herein. Alternatively, the VHH or its variant may be fused to an Fc domain, particularly a human IgGI Fc tail (also referred to herein as an IgG-fusion or IgGFc- fusion), thereby providing an Fc-fusion construct. An “Fc domain” as used herein refers to the fragment crystallizable region of an antibody, which corresponds to the tail region known to interact with cell surface Fc receptors and certain proteins of the complement system. This Fc domain is composed of two identical protein fragments derived from the second and third constant domains of the antibody’s heavy chains. While all conventional antibodies contain an Fc domain, the Fc domain fusion described herein may comprise an Fc region derived from, or a variant of, IgG, IgA, or IgD antibody Fc regions — more specifically, IgGI , lgG2, or lgG4 subclasses. The hinge region of lgG2 may be replaced by that of human IgGI to generate ISVD fusion constructs, and vice versa. Fc fusions may include linker moieties of varying lengths, as exemplified herein, though not limited to the examples provided. Additionally, Fc variants known to extend half-life may be incorporated, such as the M257Y/S259T/T261 E triple mutation (known as YTE) or the LS variant (M428L combined with N434S). These mutations enhance binding of the Fc domain to the neonatal Fc receptor (FcRn). Moreover, Fc mutants characterized by reduced Fc-mediated effector functions, such as the LALAPG mutant (SEQ ID NO: 71 ), may also be used. The Fc-fusion may confer additional desirable properties, including but not limited to extended serum half-life, improved biodistribution, enhanced or decreased effector function through enhanced or decreased Fc receptor binding, and facilitation of purification via protein A/G affinity methods.

In some embodiments, the Fc region is engineered to include “knob” and “hole” mutations that promote the preferential formation of heterodimers between two distinct Fc-containing polypeptide chains when co-expressed in a suitable host cell system (see, e.g., U.S. Pat. No. 7,695,963). This "knob-into-hole" (KiH) strategy enables the assembly of bispecific or multispecific molecules byfavoringthe pairingof two different heavy chains while disfavoring homodimerization. Hence, the ISVD-comprising modulator of this invention may be provided in the format of a knob- into-hole (KiH) fusion construct. In this configuration, the modulator consists of two polypeptides, each comprising a VHH with a distinct specificity and a constant region composed of a hinge, CH2, and CH3 domains. The constant regions of the two polypeptides are engineered with knob-into- hole mutations that promote preferential heterodimerization over homodimerization. The term “knob-into-hole” (KiH), as used herein, refersto a protein engineeringtechnologydesigned to guide the pairing of two polypeptides either in vitro or in vivo. This is achieved by introducing a protuberance ("knob") into one polypeptide and a compensatory cavity ("hole") into the other, precisely at their interface. The “knob” typically consists of one or more amino acid side chains that extend from the surface of the first polypeptide, fitting into the “hole” engineered in the second polypeptide. This geometric complementarity enhances the stability of the resulting heterodimer while disfavoring homodimer formation. The KiH technology is a well-established strategy for producing multispecific antibodies, particularly those with distinct binding domains. In the context of this invention, multispecific ISVD-based molecules may incorporate KiH mutations in their Fc domains and carry one or more distinct ISVDs fused to each Fc arm, enabling targeted allosteric modulation.

In additional embodiments, two or more allosteric modulators of the invention may simultaneously or sequentially bind to distinct oroverlappingepitopes within the same immunoinhibitory complex. Such co-binding may result in cooperative modulation of the three-dimensional structure, molecular dynamics, ligand-binding affinity, or signaling output of the complex. In some cases, binding of a first modulator may induce or stabilize a binding site for a second modulator, resulting in synergistic enhancement or suppression of immune signaling. In a further aspect, the invention relates to an isolated nucleic acid molecule, as defined herewith, encoding one or more of the allosteric modulators described herein, including, but not limited to, nucleic acid sequences specifically encoding multivalent or multispecific binding agents as defined herein. In related embodiments, the invention also encompasses a vector comprising said nucleic acid molecule, wherein the vector may be a plasmid, viral vector, phagemid, cosmid, or other suitable expression or cloning vector known in the art. Additionally, the invention encompasses host cells, including prokaryotic or eukaryotic cells, which have been transformed, transfected, or otherwise genetically modified to contain the nucleic acid molecule or vector described herein. Such host cells may be used for the expression, production, or functional analysis of the encoded ISVD, and may include, without limitation, bacterial cells, yeast cells, insect cells, plant cells, or mammalian cells, such as CHO, HEK293, or NSO cells, among others.

A further aspect of the invention relates to a pharmaceutical composition comprising the allosteric modulator as described herein, a nucleic acid encoding the allosteric modulator, or a vector containing such nucleic acid. In particular, the pharmaceutical composition is formulated to be pharmaceutically acceptable and, in various embodiments, may further include a suitable carrier, diluent, and/or stabilizer. These components are selected to ensure stability, bioavailability, and compatibility for therapeutic use, as further described herein.

In another aspect, the allosteric modulator of this disclosure, or the nucleic acid encoding said modulator, or a vector comprising such nucleic acid, or a pharmaceutical composition comprising any of the foregoing, may be used as a medicament for the prevention, treatment, or management of a disease, as defined herewith.

In one embodiment, the allosteric modulator of this invention, nucleic acid encoding said modulator, vector carrying such nucleic acid, and/or pharmaceutical composition comprising said modulator and/or nucleic acid and/or vector, is used in the treatment of an autoimmune disease, as defined herein.

In one embodiment, the allosteric modulator, nucleic acid encoding said modulator, vector carrying such nucleic acid, or pharmaceutical composition comprising said modulator and/or nucleic acid and/or vector, can be used in the treatment of systemic autoimmune diseases. Such systemic autoimmune diseases include, but are not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis, systemic sclerosis, mixed connective tissue disease, and Sjogren’s syndrome.

In another embodiment, said allosteric modulator, nucleic acid, vector, or pharmaceutical composition can be used in the treatment of organ-specific autoimmune diseases. Examples of such diseases include, but are not limited to, type 1 diabetes mellitus, Hashimoto’s thyroiditis, Graves’ disease, multiple sclerosis, autoimmune hepatitis, primary biliary cholangitis, autoimmune gastritis, autoimmune nephropathies such as IgA nephropathy and membranous nephropathy, and autoimmune encephalitis.

In a further embodiment, the allosteric modulator, nucleic acid, vector, or pharmaceutical composition is used for treating inflammatory bowel diseases, including Crohn’s disease and ulcerative colitis.

In yet another embodiment, the allosteric modulator, nucleic acid, vector, or pharmaceutical composition is employed in the treatment of autoimmune skin diseases. Such diseases include, but are not limited to, psoriasis, vitiligo, pemphigus, pemphigoid, bullous pemphigoid, and epidermolysis bullosa acquisita.

In one embodiment, the allosteric modulator, nucleic acid, vector, or pharmaceutical composition can be used in the treatment of autoimmune neurological diseases. These include multiple sclerosis, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), autoimmune encephalitis, myasthenia gravis, and autoimmune limbic encephalitis.

In another embodiment, said modulator, nucleic acid, vector, or pharmaceutical composition is used in the treatment of autoimmune hematologic diseases. Examples include autoimmune hemolytic anemia (AIHA), immune thrombocytopenia (ITP), acquired hemophilia, cold agglutinin disease, and paroxysmal nocturnal hemoglobinuria (PNH).

In further embodiments, the allosteric modulator, nucleic acid, vector, or pharmaceutical composition can be used for the treatment of vasculitis and autoimmune rheumatic diseases. Such diseases include giant cell arteritis, Takayasu’s arteritis, polyarteritis nodosa, granulomatosis with polyangiitis (Wegener’s granulomatosis), Churg-Strauss syndrome, Behpet’s disease, polymyalgia rheumatica, and adult-onset Still’s disease.

In still further embodiments, the allosteric modulator, nucleic acid, vector, or pharmaceutical composition is used in the treatment of other autoimmune disorders, including autoimmune pancreatitis, autoimmune polyendocrine syndromes (types 1 , 2, and 3), autoimmune inner ear disease, autoimmune retinopathy, autoimmune angioedema, chronic recurrent multifocal osteomyelitis (CRMO), and autoimmune autonomic ganglionopathy.

In various embodiments, the autoimmune diseases that may be treated by the allosteric modulator of this invention, nucleic acid encoding said modulator, vector carrying such nucleic acid, and/or pharmaceutical composition comprising said modulator and/or nucleic acid and/or vector may be selected from the following non-limiting list, which is intended to encompass any known or future autoimmune disease: rheumatoid arthritis, systemic lupus erythematosus (lupus), type 1 diabetes mellitus (insulin-dependent diabetes), psoriasis, multiple sclerosis, Crohn’s disease, ulcerative colitis, Hashimoto’s thyroiditis, Graves’ disease, Addison’s disease, vitiligo, pernicious anemia, celiac disease (gluten-sensitive enteropathy), Sjogren’s syndrome, myasthenia gravis, polymyalgia rheumatica, giant cell arteritis, pemphigus, pemphigoid, dermatomyositis, polymyositis, ankylosing spondylitis, Goodpasture syndrome, primary biliary cholangitis (autoimmune cholangitis), autoimmune hepatitis, idiopathic thrombocytopenic purpura (ITP), Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), sarcoidosis, granulomatosis with polyangiitis (Wegener’s granulomatosis), eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome), autoimmune pancreatitis, relapsing polychondritis, Behpet’s disease, primary sclerosing cholangitis, mixed connective tissue disease, juvenile idiopathic arthritis (juvenile rheumatoid arthritis), Takayasu’s arteritis, polyarteritis nodosa, eosinophilic esophagitis, IgA nephropathy (Berger’s disease), chronic urticaria, bullous pemphigoid, cold agglutinin disease, Raynaud’s phenomenon, chronic atrophic gastritis, paraneoplastic syndromes, membranous nephropathy, stiff person syndrome, acquired hemophilia, chronic recurrent multifocal osteomyelitis (CRMO), autoimmune inner ear disease, autoimmune retinopathy, autoimmune encephalitis, autoimmune polyendocrine syndromes types 1 , 2, and 3, chronic mucocutaneous candidiasis, pemphigoid gestationis, autoimmune hemolytic anemia (Al HA), autoimmune polyglandular syndrome, lgG4-related disease, primary autoimmune neutropenia, immune thrombocytopenia (formerly idiopathic thrombocytopenic purpura), autoimmune lymphoproliferative syndrome (ALPS), autoimmune enteropathy, autoimmune blistering diseases (including epidermolysis bullosa acquisita and linear IgA dermatosis), autoimmune chronic active hepatitis, autoimmune limbic encephalitis, paroxysmal nocturnal hemoglobinuria (PNH), autoimmune angioedema, lupus nephritis, seronegative spondyloarthropathy, adult-onset Still’s disease, chronic periaortitis, and autoimmune autonomic ganglionopathy.

In one embodiment, the allosteric modulator of this invention, nucleic acid encoding said modulator, vector carrying such nucleic acid, and/or pharmaceutical composition comprising said modulator and/or nucleic acid and/or vector, may be used in the treatment of graft-versus-host disease (GVHD), as defined herewith. This includes both acute and chronic forms of GVHD, which may arise following allogeneic hematopoietic stem cell transplantation or solid organ transplantation. In one embodiment, the allosteric modulator of this invention, nucleic acid encoding said modulator, vector carrying such nucleic acid, and/or pharmaceutical composition comprising said modulator and/or nucleic acid and/or vector, may be used in the treatment of allergic diseases, as defined herewith. Such allergic diseases include, but are not limited to, IgE-mediated immediate hypersensitivity reactions such as allergic rhinitis (hay fever), allergic asthma, atopic dermatitis (eczema), food allergies, and anaphylaxis. Additionally, this embodiment encompasses non-lgE- mediated hypersensitivity reactions, including contact dermatitis, allergic conjunctivitis, and certain forms of drug allergies.

In further embodiments, the allosteric modulator, nucleic acid, vector, or pharmaceutical composition of this invention can be used in the treatment of chronic allergic conditions characterized by sustained immune activation and inflammation, such as chronic urticaria (hives) and eosinophilic esophagitis. Furthermore, the treatment includes treatment of occupational and environmental allergies triggered by allergens such as pollen, dust mites, molds, animal dander, and insect venoms.

A further aspect relates to said allosteric modulator, nucleic acid molecule, vector, or pharmaceutical composition, as described herein, for use as a diagnostic. In a particular embodiment, kits are provided which contain means to detect said protein complex, including the allosteric modulatoror lSVDs as described herein, allowingto detect and/or modulate downstream signaling in a system, which may be an in vitro or in vivo system. It is envisaged that these kits are provided for a particular purpose, such as for modulating the protein complex response or signaling, or tor in vivo imaging, or for diagnosis of an altered protein complex quantity, response or effect in a subject. In another embodiment, said kit is provided which contains means including a nucleic acid molecule, a vector, or a composition as described herein. The means further provided by the kit will depend on the methodology used in the application, and on the purpose of the kit. For instance, detection of a labelled allosteric modulator, or nucleic acid molecule as described herein, which may be desired for protein complex quantification on nucleic acid or protein level. For protein-based detection, the kits typically contain labelled or coupled protein-complex binding agents such as said allosteric modulator and/or ISVDs described herein. Likewise, for detection at the nucleic acid level, the kits may contain labels for nucleic acids such as primers or probes. Further control agents, antibodies or nucleic acids may also be provided in the kit. A standard, for reference or comparison, a substrate or signaling component, a reporter gene or protein or other means for using the kit may also be included. Of course, the kit may further comprise pharmaceutically acceptable excipients, buffers, vehicles or delivery means, an instruction manual and so on.

Another aspect of the invention provides for a method for detecting the presence, absence or level of protein complex comprising at least one immunoinhibitory surface receptor and at least one ligand in a sample, the method comprising: contacting the sample with the allosteric modulator, binding agent or ISVD as described herein, and detecting the presence or absence or level, i.e. quantifying, the bound ISVD, which is optionally a labelled, conjugated or multispecific binding agent. The sample used herein may be a sample isolated from the body, such as a body fluid, including blood, serum, cerebrospinal fluid, among others, or may be an extract, such as a protein extract, a cell lysate, etc.

Furthermore, the allosteric modulatoror bindingagent, in particularcomprisinga protein-complex- specific ISVD, the nucleic acid molecule, the vector, or the pharmaceutical composition comprising said binding agent, as described herein may also be used for in vivo imaging.

For the purpose of detection and/or imaging, in vitro or in vivo, the binding agent, comprising a protein complex-specific ISVD, as described herein may further comprise in some embodiments a detection agent, such as a tag or a label. For instance, the ISVDs, VHHs, or Nbs as exemplified herein were also tagged, by the 6-His-EPEA double tag. Such a tag allows affinity purification and detection of the antibody or active antibody fragments of the invention.

Some embodiments comprise the allosteric modulator or binding agent, or the protein-complex specific ISVD, further comprising a label or tag, or more specifically, the binding agent labelled with a detectable marker. The term detectable label or tag, as used herein, refers to detectable labels or tags allowing the detection and/or quantification of the allosteric modulator or binding agent as described herein, and is meant to include any labels/tags known in the art for these purposes. Particularly preferred, but not limiting, are affinity tags, such as chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), poly(His) (e.g., 6x His or His6), biotin or streptavidin, such as Strep-tag®, Strep-tag II® and Twin-Strep-tag®; solubilizing tags, such as thioredoxin (TRX), poly(NANP) and SUMO; chromatography tags, such as a FLAG-tag; epitope tags, such as V5-tag, myc-tag and HA-tag; fluorescent labels or tags (i.e., fluorochromes/-phores), such as fluorescent proteins (e.g., GFP, YFP, RFP etc.) and fluorescent dyes (e.g., FITC, TRITC, coumarin and cyanine); luminescent labels or tags, such as luciferase, bioluminescent or chemiluminescent compounds (such as luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate ester, dioxetane or GFP and its analogs); phosphorescent labels; a metal chelator; and (other) enzymatic labels (e.g., peroxidase, alkaline phosphatase, beta-galactosidase, urease or glucose oxidase); radioisotopes. Also included are combinations of any of the foregoing labels or tags. Technologies for generating labelled polypeptides and proteins are well known in the art. An allosteric modulator or binding agent comprising a protein complex-specific ISVD of the invention, coupled to, or further comprising a label or tag allows for instance immune-based detection of said bound protein- complex-specific agent. Immune-based detection is well known in the art and can be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as described above. See, for example, U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241 . In the case where multiple antibodies are reacted with a single array, each antibody can be labelled with a distinct label or tag for simultaneous detection. Yet another embodiment may comprise the introduction of one or more detectable labels or other signal-generating groups or moieties, or tags, depending on the intended use of the labelled or tagged allosteric modulator or binding agent of the present invention. Other suitable labelswill be clear to the skilled person, and for example include moieties that can be detected using NMR or ESR spectroscopy. Such labelled allosteric modulators, such as immunoinhibitory receptor-ligand protein complex-specific ISVDs or Nanobodies as described herein may for example be used for /n vitro, in vivo or in situ assays (including immunoassays known per se such as ELISA, RIA, EIA and other "sandwich assays", etc.) as well as in vivo imaging purposes, depending on the choice of the specific label.

So, in another aspect, an in vitro method is disclosed for detection of the localization and distribution of said protein complex of interest in a biological sample, comprising the steps of: reacting the sample with a binding agent disclosed herewith, comprising a immunoinhibitory receptor-ligand protein complex-specific ISVD as described herein, and detecting, the localization and distribution of said protein complex binding in said biological sample. The biological sample as used herein may envisage any sample derived from a biological system, and for example comprise cells of healthy or cancerous tissue, or an extract or an in vitro sample, or a body fluid such as cerebrospinal fluid or blood.

Said method may be particularly useful for profiling immunoinhibitory complexes, such as hPD- 1 »hPD-L1 , hPD-1 »hPD-L2, CTLA-4»CD80/CD86, and TIGIT’PVR, among others, in tumor microenvironments, stromal compartments, or associated lymphoid tissues. The ability to detect and spatially resolve these complexes may allow for the assessment of the immune landscape of a tumor, evaluation of the degree of immune evasion, and stratification of patients for immunotherapeutic regimens targeting specific inhibitory pathways. The method involves obtaining a biological sample, such as a tumor biopsy, fine needle aspirate, or surgically resected tissue section, from a subject suspected of or diagnosed with cancer. Cancer types in which this approach is particularly relevant include, but are not limited to, non-small cell lung carcinoma (NSCLC), melanoma, renal cell carcinoma (RCC), urothelial carcinoma, head and neck squamous cell carcinoma (HNSCC), gastric cancer, hepatocellular carcinoma (HCC), colorectal cancer (CRC), triple-negative breast cancer (TNBC), and various hematologic malignancies such as classical Hodgkin lymphoma (cHL), non-Hodgkin lymphoma (NHL), and certain leukemias including chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). Each of these malignancies may display distinct patterns of immune checkpoint expression and engagement, which can serve as biomarkers of immune suppression or immune escape. Detection of the protein complex may be achieved by immunohistochemistry (IHC), immunofluorescence (IF), or proximity ligation assay (PLA), the modulators of this disclosure that are specific for conformational epitopes unique to the complex, rather than its unbound components. For example, detection of the hPD- 1 »hPD-L1 complex, but not free PD-1 or PD-L1 , can be enabled through the use of conformationspecific binders such as the allosteric modulators (PAMs) disclosed herein, which selectively recognize the receptor-ligand complex in its native state. Labeling these binders with fluorescent tags, enzymes (e.g., HRP), or oligonucleotide conjugates allows for multiplexed imaging or signal amplification. The method may further comprise the use of digital pathology and image analysis algorithms to quantify signal intensity and localization at single-cell resolution, enabling scoring systems based on the density of complex-positive cells within tumor nests versus immune- infiltrated regions. These data can be integrated with conventional histopathology and other biomarkers such as tumor mutational burden (TMB), microsatellite instability (MSI), or gene expression signatures (e.g., interferon-y response), thereby providing a multidimensional immune profile. Stratification of patients based on the abundance, localization, or pattern of immune inhibitory complex formation can inform therapeutic decisions. For instance, patients whose tumors exhibit high levels of PD-1 »PD-L1 complex formation at the interface of T cells and tumor cells may be more responsive to anti-PD-1 or anti-PD-L1 therapies.

In another aspect, the disclosure provides a method for producing an allosteric modulator of a protein complex comprising at least one inhibitory immune receptor and at least one ligand. In a preferred embodiment, the method comprises the steps of: selecting one or more modulators that specifically bind to an epitope predominantly present on the target protein complex, as defined herein; further screening or identifying from the selected modulators those that induce positive cooperativity within the protein complex, as defined herein; and optionally, identifying among the selected allosteric modulators those capable of modulating downstream signaling pathways mediated by one or more proteins of the immunoinhibitory complex. In various embodiments, the selection and characterization of allosteric modulators may be performed using a comprehensive range of binding and functional assays designed to detect, quantify, and/or evaluate specific interactions with the target protein complex. It will be appreciated that, for this purpose, a unique label or tag is often employed, such as a peptide label, nucleic acid label, chemical label, fluorescent label, or radio frequency tag, as further described herein.

Various methods may be used to determine specific binding (as defined hereinbefore) between the allosteric modulator obtained according to the method of this invention and its target protein complex. Commonly employed binding assays include surface plasmon resonance (SPR) and biolayer interferometry (BLI), which provide real-time, label-free analysis of binding kinetics and affinity. Enzyme-linked immunosorbent assays (ELISA) can also be utilized to measure the binding specificity and strength through antibody-antigen recognition. Additional biophysical techniques such as fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), and microscale thermophoresis (MST) are valuable for monitoring binding events and conformational changes within protein complexes. Co-immunoprecipitation (Co-IP) and pull-down assays may further verify physical interactions in complex biological samples such as cell lysates. All such methods are standard in the field and well known to those skilled in the art, as described, for example, in "Molecular Cloning: A Laboratory Manual", 4^th edition (2012), by Green MR and Sambrook J³¹, and are further exemplified in the Examples section of this disclosure.

Positive cooperativity induced by the allosteric modulators obtained through the method of this invention can be assessed through biophysical or biochemical methods that detect changes in binding affinity or kinetics upon modulator binding, as known in the art. Techniques such as FRET, isothermal titration calorimetry (ITC), and analytical ultracentrifugation may provide detailed insights into cooperative binding behavior. From these data, quantitative parameters including the cooperativity factor a (alpha) and the Hill coefficient (n_H) can be derived, offering complementary measures of heterotropic and optionally homotropic cooperative interactions, respectively, within the protein complex. Beyond bindingand cooperativity studies, pharmacological assays measuring receptor signaling and downstream cellular responses may be used for evaluating the functional modulation of immune complexes by allosteric modulators. These assays may include reporter gene assays, calcium flux measurements, and/or phosphorylation status analysis via western blot or flow cytometry, among other techniques known to the skilled person. From these assays, key pharmacological parameters such as the EC₅₀ (half maximal effective concentration) can be derived. The EC₅₀ represents the concentration of the allosteric modulator required to elicit 50% of its maximal biological effect, serving as an important measure of the modulator’s potency. In various embodiments, the effects of the allosteric modulators described herein on downstream signaling mediated by proteins of the target complex may be assessed using cellular assays. The cell types used to evaluate allosteric modulators of immune receptor complexes can be primary cells, immortalized cell lines, and/or genetically engineered cells. Primary cells are freshly isolated from tissues or blood and include peripheral blood mononuclear cells (PBMCs), T cells, B cells, dendritic cells, macrophages, and natural killer (NK) cells. Immortalized cell lines, such as JurkatT cells, Raji B cells, and THP-1 monocytes, are widely used for their ease of culture and reproducibility. Additionally, cells can be genetically engineered to express specific immune receptors or ligands of interest. Functional assays for characterizing the allosteric modulators of this invention may utilize co-cultures of relevant cell types to better mimic physiological interactions. For example, the mixed lymphocyte reaction (MLR) is a classic assay where primary T cells from one donor are co-cultured with antigen-presenting cells (APCs), such as dendritic cells, from a genetically different donor or donors¹⁵⁴. This interaction stimulates T cell activation and proliferation through immune synapse formation. The MLR and similar co-culture systems may provide relevant models to assess the effects of the allosteric modulators of this disclosure on inhibitory immune receptor-ligand complexes, including signaling mediated by these complexes, in a controlled yet physiologically meaningful setting.

In various embodiments, the method for producing the allosteric modulator of this invention involves a selection procedure utilizing immune libraries combined with multiple successive biopanning steps to enrich candidate modulators (binders). Preferably, these immune libraries are derived from Camelidae species, such as llama immune libraries, which are well-known sources of single-domain antibodies (Nanobodies). The candidate binder display platforms may include, but are not limited to, phage display, bacteriophage display, mammalian cell display, or other display technologies recognized in the art. In a particularly preferred embodiment, phage display is employed as the candidate binder display system. In another preferred embodiment, mammalian display is employed as the candidate binder display system. The successive biopanning steps are specifically designed to select binders that recognize and bind to the receptor-ligand complex - for example, the PD-1 »PD-L1 complex - rather than to isolated (unbound) receptor or ligand molecules. This approach enables the identification of modulators that specifically target conformational epitopes presented by the assembled immune complex.

In a preferred embodiment, the multiple successive biopanning steps may comprise: a) a step wherein the candidate library is incubated with an immobilized complex constituent, such as a receptor or receptors, in the presence of an excess of a different soluble complex subunit, such as a ligand or ligands of said receptor(s), and b) a subsequent step wherein the candidate library selected for binding to the immobilized fraction in a) is used as input and incubated with a soluble complex constituent which was immobilized in a), along with an immobilized complex subunit that was present in solution in a), and c) a step wherein the candidate library selected for binding to the immobilized fraction in b) is used as input and incubated with an immobilized complex subunit which was immobilized in a) and a soluble complex constituent which was immobilized in a), and d) a subsequent step wherein the candidate library selected for binding to the immobilized fraction in c) is used as input and incubated with a soluble complex constituent which was immobilized in a) and an immobilized complex subunit which was present in solution in a).

In this scenario, each round progressively enriches for binders that target allosteric epitopes of the receptor-ligand complex while simultaneously counter-selecting binders that bind to the excess soluble protomer or subunit. In a related embodiment of the herein described selection method, the complex subunits are immobilized on an ELISA plate that has been pre-coated with a capture reagent, such as streptavidin or its variants/derivatives, as known in the field. Furthermore, at least one of the individual members of the protein complex is distinguishably labeled. This labeling facilitates the detection and separation via ELISA of phages or cells displaying binding agents that bind to the immobilized fraction, ultimately enabling identification of phages or cells that display binding agents specifically targeting the conformational epitopes of the protein complex rather than its individual subunits.

In various specific embodiments, the method includes elution and amplification of selected phages after a selection round. Said phage amplification may be performed in a suitable host, such as a bacterial host. In a particular embodiment, phage amplification is performed in Escherichia coli. Alternatively, different display methods may be applied for the panning such as yeast display, or mammalian display.

In various embodiments, the method for producing allosteric modulators of immune receptorligand complexes encompasses identification of modulators that affect cooperativity within said complexes. In a preferred embodiment, the identified binder induces positive cooperativity characterized by a cooperativity factor a (alpha) greater than 1 (a > 1 ) within the complex it binds to. Positive cooperativity, as defined herein, may be determined by comparing the equilibrium dissociation constants (K_D) of the protein complex in the absence and presence of the allosteric modulator, where a decrease in K_D upon modulator binding indicates enhanced binding affinity and thus positive heterotropic cooperativity. Additionally, in further embodiments, the modulation of cooperativity by the binder may also be assessed by determining the Hill coefficient (n_H), which serves as a measure of homotropic cooperativity within multimeric protein complexes that contain multiple identical ligand-binding sites. A Hill coefficient greater than 1 (n_H > 1 ) indicates that the binding of one ligand molecule increases the affinity or likelihood of subsequent ligand molecules binding to other identical sites. To comprehensively evaluate these cooperative binding phenomena, a variety of biophysical and biochemical methods may be employed, such as surface plasmon resonance (SPR), bio-layer interferometry (BLI), fluorescence resonance energy transfer (FRET), isothermal titration calorimetry (ITC), microscale thermophoresis (MST), or analytical ultracentrifugation, among others.

In additional embodiments, the method disclosed herewith may include one or more supplementary steps to further characterize, optimize, and/or develop the properties of the candidate allosteric modulator. Such steps may include, without limitation, affinity maturation, either through directed evolution, site-directed mutagenesis, or library-based selection techniques; expression of the optimized orselected amino acid sequence in a suitable host system (e.g., E. coli, CHO cells, yeast, or insect cells, among others); screeningfor binding and/or activity against the target antigen, including both the protein complex and its individual components; determination of the amino acid or nucleotide sequence of selected modulators by sequencing and/or mass spectrometry, among other techniques; and/or humanization, wherein one or more amino acid substitutions are introduced to reduce immunogenicity while retaining binding specificity and activity.

Furthermore, in various embodiments, the allosteric modulator obtained by the method of this invention may be formatted into multivalent constructs, such as tandem repeats of a binding domain targeting the same epitope (homo-valent) or different epitopes within the same or distinct immunoinhibitory complexes (hetero-valent). For example, trivalent or tetravalent modulators may enhance binding to clustered immune complexes on cell surfaces, improving both the potency and specificity.

In further related embodiments, the allosteric modulator obtained by the method of this invention may be formatted into multispecific constructs, in which binding domains specific to distinct epitopes are incorporated into a single molecular scaffold. Such formats may include, without limitation, bispecific constructs (e.g., IgG-like formats, diabodies, dual-variable domain constructs), trispecific constructs, tetraspecific constructs, and/or fusion proteins, such as Fc fusions.

In a further embodiment, the method disclosed herein is employed to identify an allosteric modulatorthat selectively binds to a conformational epitope predominantly present on the target immunoinhibitory complex. Such conformational epitopes are three-dimensional structures formed by the spatial arrangement of amino acid residues across one or more protein subunits within the complex, and are distinct from linear epitopes found on isolated subunits. The allosteric modulator or binder may recognize conformational epitopes located on: i) all subunits of the complex, ii) a single subunitwithin the complex, oriii) a subset of the subunits forming the complex, depending on the nature and stoichiometry of the immune receptor-ligand assembly.

For example, in a binary complex such as PD-1 »PD-L1 , the allosteric modulator may bind to a conformational epitope formed jointly by residues from both PD-1 and PD-L1 (i.e., spanning both subunits), or alternatively, it may recognize an epitope localized entirely on PD-1 or solely on PD- L1. Similarly, in a ternary complex such as BTLA»HVEM»CD160, the modulator could bind to an epitope involving residues from all three proteins simultaneously, or it may target an epitope present on any two of these subunits (e.g., BTLA-HVEM interface), or it may bind specifically to a conformational epitope unique to a single subunit within the trimeric assembly. Extending this concept to higher-order complexes, such as the quaternary CTLA-4»CTLA-4»B7-1 »B7-1 complex, the allosteric modulator may interact with conformational epitopes encompassing all four subunits, or subsets thereof - such as epitopes formed by both CTLA-4 molecules, by one CTLA-4 and one B7-1 molecule, or by any single subunit independently.

Therefore, it shall be understood that the method disclosed herein allows for the identification of allosteric modulators that bind to conformational epitopes distributed across all constituents, multiple (but not all) constituents, or individual subunits of the target immunoinhibitory complex.

In a preferred embodiment, the candidate allosteric modulators undergoing selection according to the method of this invention are provided as a library of antibodies or active antibody fragments, preferably single domain antibodies or immunoglobulin single variable domains (ISVDs). More preferably, the candidate modulators are VHHs, derived from the heavy-chain-only antibodies of Camelidae species. These libraries may be generated using immune, semi-synthetic, or synthetic approaches, but are preferably obtained through immunization of a suitable host animal, such as a llama or alpaca, with a cross-linked receptor-ligand antigen complex. The cross-linked immunogen is designed to preserve and present the native quaternary structure and conformational epitopes of the immune inhibitory complex (e.g., PD-1 »PD-L1 , BTLA»HVEM, CTLA- 4»B7-1 ), thereby promoting the generation of binders that selectively recognize epitopes predominantly present on the complex. In certain embodiments, the immunogen format may be prepared according to the Chill & Disco method referenced herein earlier (EP3194976), or a comparable technique, wherein the receptor-ligand complex is stabilized (e.g., by chemical cross- linking, engineered disulfide bridges, or fusion constructs), purified, and used as the immunogen for library generation.

Immunization methods

In various embodiments, the collection of candidate modulators of the method disclosed herewith is a library of antibodies, active antibody fragments, immunoglobulin single variable domains (ISVDs), single domain antibodies, VHHs, or Nanobodies (Nbs), all of which are defined in this application.

While any assortment of binding agents could theoretically include conformation-selective modulators targeting immune protein complexes, a favored approach involves generating an immune library of allosteric modulators, specifically antibodies or antibody fragments, such as ISVDs, single domain antibodies, VHHs, or Nbs. Such immune libraries of allosteric modulators of this invention may be obtained by immunizing an animal with a cross-linked protein complex, to stimulate the animal's immune system and expose it to the unique conformational epitopes present in the complex. In a preferred embodiment, an immune library comprising single variable domains of immunoglobulins, as defined herein, is generated.

In theory, animals may be immunized with mixtures of interacting components of a protein complex. However, due to the typically short half-life of many such complexes - particularly transient protein-protein interactions (PPIs), which may persist for only 0.1 to 1 second - it is generally preferable to stabilize these transient complexes, for example, by chemically crosslinking the individual interacting components prior to immunization. This approach allows animals to be immunized with antigens that mimic the covalent association of the transient complex, thereby eliciting and maturing immunoglobulins that bind to the conformational epitopes of these transient complexes. Methods for cross-linking protein-protein complexes are well-established and can be performed using various techniques known to those skilled in the art. Importantly, these reactions must occur under conditions that preserve the native conformation and functionality of the complex. Detailed protocols and reagents suitable for such applications can be found in several sources, including but not limited to: Leitner et al., 201 O'⁵⁵ and Bich etaL, 2010'⁵⁶, both of which are incorporated here by reference.

Chemical cross-linking reagents used in the field are classified based on their reactivity, such as amine- or thiol-reactive, and whether they are homo- or heterobifunctional. These reagents typically consist of two reactive sites connected by a spacer or linker, often an alkyl chain. The primary targets for these cross-linkers are lysine residues and protein N termini, with N- hydroxysuccinimidyl or sulfosuccinimidyl esters being the preferred target groups. Common examples of cross-linking reagents include disuccinimidyl suberate (DSS) and disuccinimidyl glutarate (DSG), as well as their sulfo analogs bis(sulfosuccinimidyl) suberate (BS3) and bis(sulfosuccinimidyl) glutarate. Lysine-specific cross-linking is advantageous due to the abundance of lysine residues in proteins (approximately 6%) and high reaction specificity. Crosslinkingcan also target cysteine residues using maleimides, though the lower abundance of cysteine (<2%) makes this approach less common. Other cross-linking methods include arginine-specific cross-linking and acidic residue cross-linking¹⁵⁷’¹⁵⁸. Moreover, glutaraldehyde, a widely used homobifunctional cross-linker, is reactive with primary amines and is commonly employed in biochemical applications to covalently stabilize protein complexes. Due to its ability to form Schiff base linkages between lysine residues or N-terminal amine groups, glutaraldehyde is particularly effective in cross-linking proteins at their native interaction interfaces, making it suitable for preservingthe conformational integrity of multimeric protein complexes such as immune receptorligand assemblies. Additionally, heterobifunctional cross-linkers incorporate different reactive groups, such as lysine- and cysteine-reactive functionalities, or combine chemical and photoinduced cross-linking mechanisms. Formaldehyde, despite its lower specificity, can also serve as a cross-linker, primarily targeting lysine and tryptophan residues. These reagents and their applications are extensively reviewed in the literature, including studies by Sutherland et al., 2008'⁵⁹ and Toews etal., 201 (T⁶⁰.

Functionalized cross-linking reagents encompass a diverse array of chemical activities that enable both covalent stabilization of biomolecular and higher-order complexes and downstream analytical applications. These functionalities may include, but are not limited to, stable isotope labels (e.g., for quantitative proteomics), affinity tags (such as biotin or FLAG), photoactivatable groups, cleavable linkers (e.g., disulfide or MS-cleavable), or reporter moieties that generate characteristic fragmentation signatures in tandem mass spectrometry (MS/MS) analyses. Such features facilitate precise identification, enrichment, or quantification of cross-linked peptides or complexes in structural and interaction studies. For example, commercially available cross-linking reagents such as DSS or BS3, supplied by companies like Creative Molecules and Thermo Scientific’s Pierce division, often incorporate stable isotopes. More advanced variants of these reagents have also been synthesized in labeled forms to enhance analytical capabilities. Additionally, affinity-tagged cross-linkers commonly include biotin as the affinity moiety, facilitating the enrichment and isolation of modified peptides through avidin affinity chromatography. Another example includes azide-containing cross-linking reagents, which facilitate selective enrichment of azide-containing peptides. Yet another category of functionalized reagents employs linkers with designed fragmentation properties, often containing labile bonds that undergo facile cleavage during collision-induced dissociation. These reagents are widely used for studying protein- protein interactions and topologies using chemical cross-linking coupled with mass spectrometry.

Methods of immunization are well-established in the field. To immunize an animal with a crosslinked protein complex, the complex or its individual protomers can first be produced and purified using conventional recombinant protein expression and purification techniques. Typically, this involves expressing the protein or protein complex in a suitable host cell system, followed by purification using standard approaches such as affinity chromatography, including tag-based purification, or antibody-based purification methods targeting specific epitopes or tags. In specific embodiments, the baculovirus-insect cell expression system (e.g., Sf9 cells) may be employed due to its ability to produce properly folded, post-translationally modified eukaryotic proteins, which is often criticalfor preserving native conformation and complexassembly. However, otherexpression systems, including bacterial (e.g., E. coli), yeast (e.g., Pichia pastoris), and mammalian cell lines (e.g., HEK293, CHO), are also viable and may be selected depending on the protein complexity, post-translational modification requirements, and yield considerations.

Methods for purifying protein complexes containing membrane proteins are detailed in various literature sources, including Eroglu et al., 2002¹⁶¹ , Kashino, 2003¹⁶², Daulat et al., 2007¹⁶³, Smith, 2016¹⁶⁴, among others. These membrane protein complexes may be reconstituted in phospholipid vesicles^165-167. Forsoluble proteins, methodsfor recombinant expression and purification of protein complexes are well-documented. Other immunization approaches include using whole cells expressing a protein complex or fractions thereof, immunization with viruses or virus-like particles expressing the protein complex of interest, as described in patent documents such as WO2010070145 and WO2011083141 .

The selection of immunization techniques depends on generating an appropriate immune response suitable for the desired applications. In this line, various animals can be used for immunization with cross-linked protein complexes. In a preferred embodiment, the allosteric modulator of this invention is obtained through immunization of a mammal belonging to the family Camelidae, such as a camel or llama.

In one aspect, the method described herein involves displaying a collection of candidate allosteric modulators, preferably an immune library, on the extracellular surface of a population of cells. Various suitable surface display methods are reviewed in Hoogenboom, 2005'⁶³ and Zhou et al., 2006'⁶⁹, and include phage display, bacterial display, yeast display, and mammalian display. In one embodiment, the candidate allosteric modulators are displayed on the cell surface of yeast cells. Any method capable of displaying a protein on the surface of yeast is encompassed by the present disclosure. Different yeast surface display techniques securely link each binding agent encoded by the library to the extracellular surface of the yeast cell containing the plasmid encoding that protein. Most yeast display methods described to date utilize Saccharomyces cerevisiae, but other yeast species, such as Pichia pastors, could also be employed. Specifically, in some embodiments, the yeast strain may belong to genera like Saccharomyces, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces, Yarrowia, and Candida.

Within these genera, yeast species such as S. cerevisiae, P. pastoris, H. polymorpha, S. pombe, K. lactis, Y. lipolytica, and C. albicans are suitable candidates. Systems of yeast expression of fusion proteins typically involve anchor proteins which facilitate surface expression of cell-surface proteins. Hence, in some embodiments, the protein variants or complexes intended for display are genetically fused to a GPI anchor protein. The most commonly utilized yeast display system is the commercialized GPI protein alpha-agglutinin system¹⁷⁰ which comprises two subunits, Aga1 p and Aga2p. In this system, heterologous proteins are fused to the C-terminus of Aga2p. Aga2p forms a complex with Aga1 p via disulfide bonds, and Aga1 p is anchored to the yeast surface through a GPI (Glycosyl-Phosphatidyl-lnositol) anchor. This arrangement ensures stable and effective display of the fused proteins on the surface of yeast cells. Examples of other suitable GPI anchor proteins include flocculin proteins such as Flo1 , as well as Sed1 , Cwp1 , Cwp2, Tipi , and Tir1/Srp1. In various embodiments, the anchor protein is selected from a group including modified GPI anchor variants, and major cell wall proteins like CCW14, CIS3, CWP1 , PIR1 , and PIR3.

In specific embodiments, a method involving in vivo biotinylation of the protein to be displayed is utilized. Here, the protein is genetically fused to a yeast secretory protein and a biotin-acceptor peptide (BAP). A tag such as HA or FLAG® is engineered downstream of the protein variant sequence. Common secretory proteins include yeast alpha mating factor prepro 1 (WTaMFpp), invertase leader, synthetic leaders, and engineered alpha mating factor prepro aMFpp8. The fusion gene may be controlled by an inducible promoter like the galactose-inducible GAL1 -10 promoter. Before inducing protein expression, the outer surface of yeast cells is chemically conjugated to avidin. Upon induction, the biotin-acceptor peptide (BAP) within the fusion protein is intracellularly biotinylated by a co-expressed biotin ligase, such as BirA. The biotinylated fusion protein is secreted and captured on the yeast cell surface by avidin due to the high-affinity avidin-biotin interaction. The methods described here can also be implemented using prokaryotic/bacterial host cells. Therefore, in certain embodiments, the host cell utilized in protein display is a prokaryotic/bacterial cell. Prokaryotic/bacterial cell-surface display allows peptides and proteins to be presented on the surface of microbial cells by fusing them with anchoring motifs. The protein to be displayed, known as the passenger protein, can be fused to an anchoring motif, the carrier protein, through N- terminal fusion, C-terminal fusion, or sandwich fusion. The efficiency of surface display is influenced by the characteristics of the carrier protein, passenger protein, host cell, and the fusion method used¹⁷¹.

Prokaryotic host cells suitable for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia (e.g., E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., S typhimurium), Serratia (e.g., S. marcescans), Shigella, Bacilli, such as B. subtilis and B. licheniformis (e.g., B. licheniformnis 41 P disclosed in DD266710), Pseudomonas (such as P. aeruginosa), and Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31 ,446), although other strains, such as E. coli B, E. coli X1776 (ATCC 31 ,537), and E. coli W3110 (ATCC 27,325), are also suitable. These examples illustrate the non-limiting diversity of prokaryotic hosts available for implementing the methods described herein.

When the host cell is a prokaryotic cell, suitable cell surface anchor proteins include bacterial outer membrane proteins such as pili, flagella, lipoproteins, ice nucleation proteins, and autotransporters. Exemplary bacterial proteins used for heterologous protein display include LamB¹⁷², OmpA¹⁷³, and intimin¹⁷⁴. Additional exemplary outer membrane proteins include, but are not limited to, FliC, pullulanase, OprF, Oprl, PhoE, MisL, and cytolysin. Furthermore, in certain embodiments, the anchor protein is an artificial sequence that is assembled into, or attaches to, the outer surface of the bacterial cell.

It is also envisioned that the protein display methods utilized in the methods of this disclosure can be performed using mammalian host cells. Examples of suitable mammalian host cell lines include, without limitation: (i) Chinese hamster ovary cells (CHO), including CHOK1 cells (ATCC CCL61), DXB-11 , DG-44, and CHO cells engineered for controlled fucosylation¹⁷⁵, (ii) Monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651 ), (iii) Human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture¹⁷⁶), (iv) Baby hamster kidney cells (BHK, ATCC CCL 10), (v) Mouse Sertoli cells (TM4¹⁷⁷), (vi) African green monkey kidney cells (VERO- 76, ATCC CRL-1587), (vii) Human cervical carcinoma cells (HELA, ATCC CCL 2), (viii) Canine kidney cells (MDCK, ATCC CCL 34), (ix) Buffalo rat liver cells (BRL 3A, ATCC CRL 1442), (x) Human lung cells (W138, ATCC CCL 75), ( xi)Human liver cells (Hep G2, HB 8065), (xii) Mouse mammary tumor cells (MMT 060562, ATCC CCL51), (xiii) Testicular TR1 cells¹⁷⁸, (xiv) MRC 5 cells, (xv) FS4 cells, (xvi) Human hepatoma line (HepG2).

When utilizing mammalian cells as host cells, various portions of cell surface proteins can be employed to retain the ability to display proteins on the cell surface. Examples include the transmembrane domains of known cell membrane proteins, such as the PDGFR transmembrane domain, which has been utilized in cell surface display applications^{179 180}. Additionally, polypeptides containingGPI anchor sequences, like those derived from human decay-accelerating factor, are suitable for anchoring proteins to the cell surface¹⁸¹. Non-cleavable type II signal anchor sequences and fusion of capture molecules (such as antibodies or proteins) to membrane anchor sequences are also viable approaches (US7125973; US6919183). In some embodiments, artificial cell surface anchor sequences can be assembled into or attached to the cell membrane of mammalian cells, providing flexibility in designing most suitable systems for protein display on mammalian cell surfaces.

Further compositions and formatting of the allosteric modulators

The conformation-selective binding agents of this invention may also be modified and/or may comprise (or can be fused to) other moieties. Examples of modifications, as well as examples of amino acid residues within the binding agent of the invention that can be modified (i.e. either on the protein backbone but preferably on a side chain), methods and techniques that can be used to introduce such modifications and the potential uses and advantages of such modifications will be clear to the skilled person. For example, such a modification may involve the introduction (e.g. by covalent linking or in another suitable manner) of one or more functional groups, residues or moieties into or onto the binding agent. Examples of such functional groups and of techniques for introducing them will be clear to the skilled person, and can generally comprise all functional groups and techniques mentioned in the art as well as the functional groups and techniques known per se for the modification of pharmaceutical proteins, and in particular for the modification of antibodies or antibody fragments (including ScFv and single domain antibodies), for which reference is for example made to “Remington: The Science and Practice of Pharmacy”, 23^rd Edition (2020), edited by Adeboye A¹⁸². Such functional groups may be linked directly (for example covalently) to the binding agent, or optionally via a suitable linker or spacer, as will again be clear to the skilled person.

In the case where a binding agent shows potential therapeutic value, one of the most commonly used methods to enhance its half-life and reduce immunogenicity in pharmaceutical proteins involves attaching a suitable pharmacologically acceptable polymer, such as polyethylene glycol) (PEG) or its derivatives (e.g., methoxypoly(ethylene glycol) or mPEG). Various forms of pegylation, similar to those utilized in antibody and antibody fragment engineering (including single-domain antibodies and ScFv), can be applied^183-185. Preferably, site-directed pegylation via a cysteine residue is employed¹⁸⁶. This may involve attaching PEG to a naturally occurring cysteine residue in the binding agent, modifying the binding agent to introduce one or more cysteine residues for PEG attachment, or fusing an amino acid sequence containing cysteine residues for PEG attachment to the N- and/or C-terminus of the binding agent. These techniques are well-known to those skilled in protein engineering.

For binding agents in this invention, PEGs with molecular weights exceeding 5000 are preferred, such as more than 10,000 and less than 200,000, particularly in the range of 20,000-80,000. Less commonly used modifications to increase binding agent half-life include N-linked or O-linked glycosylation, which occur as part of co-translational and/or post-translational modifications depending on the host cell used for expressing the immunoglobulin single variable domain or polypeptide in question.

Another strategy to prolong binding agent half-life involves engineering bifunctional constructs, such as combining one Nanobody against the target immunoinhibitory complex with another against a serum protein like albumin, or fusing binding agents with peptides, such as a peptide against serum albumin. Another modification comprises N-linked or O-linked glycosylation, usually as part of co-translational and/or post-translational modification, depending on the host cell used for expressing the selected binding agents.

Yet another modification may comprise the introduction of one or more detectable labels or other signal-generating groups or moieties, depending on the intended use of the labeled binding agent. Suitable labels and techniques for attaching, using and detecting them will be clear to the skilled person, and for example include, but are not limited to, fluorescent labels, (such as IRDye800, VivoTag800, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine and fluorescent metals such as Eu or others metals from the lanthanide series), phosphorescent labels, chemiluminescent labels or bioluminescent labels (such as luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate ester, dioxetane or GFP and its analogs), radio-isotopes, metals, metals chelates or metallic cations or other metals or metallic cations that are particularly suited for use in in vivo, in vitro or in situ diagnosis and imaging, as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcal nuclease, delta- V- steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, biotinavidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholine esterase). Other suitable labels will be clear to the skilled person, and for example include moieties that can be detected using NMR or ESR spectroscopy. Such labeled binding agents of the invention may for example be used for in vitro, in vivo or in situ assays (including immunoassays known per se such as ELISA, RIA, EIA and other "sandwich assays", etc.) as well as in vivo diagnostic and imaging purposes, depending on the choice of the specific label. As will be clear to the skilled person, another modification may involve the introduction of a chelating group, for example to chelate one of the metals or metallic cations referred to above. Suitable chelating groups for example include, without limitation, 2,2',2"-(10-(2- ((2,5-dioxopyrrolidin-1 -yl)oxy)-2-oxoethyl)-1 ,4,7,10-tetraazacyclododecane-1 ,4,7-triyl)triacetic acid (DOTA), 2,2'-(7-(2-((2,5-dioxopyrrolidin-1 -yl)oxy)-2-oxoethyl)-1 ,4,7-triazonane-1 ,4- diyl)diacetic acid (NOTA), diethyl-enetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Yet another modification may comprise the introduction of a functional group that is one part of a specific binding pair, such as the biotin-(strept)avidin binding pair. Such a functional group may be used to link the binding agent to another protein, polypeptide or chemical compound that is bound to the other half of the binding pair, i.e. through formation of the binding pair. For example, a binding agent of the invention may be conjugated to biotin, and linked to another protein, polypeptide, compound or carrier conjugated to avidin or streptavidin. For example, such a conjugated binding agent may be used as a reporter, for example in a diagnostic system where a detectable signal-producing agent is conjugated to avidin or streptavidin. Such binding pairs may for example also be used to bind the binding agent of the invention to a carrier, including carriers suitable for pharmaceutical purposes. One non-limiting example is the liposomal formulations described by Cao and Suresh, 2000'^a7. Such binding pairs may also be used to link a therapeutically active agent to the binding agent of the invention.

In case binding agents are modified by linking particular functional groups, residues or moieties (as described hereinabove) to the binding agent, then often linker molecules will be used. Preferred “linker molecules” or “linkers” are peptides of 1 to 200 amino acids length, and are typically, but not necessarily, chosen or designed to be unstructured and flexible. For instance, one can choose amino acids that form no particular secondary structure. Or, amino acids can be chosen so that they do not form a stable tertiary structure. Or, the amino acid linkers may form a random coil. Such linkers include, but are not limited to, synthetic peptides rich in Gly, Ser, Thr, Gin, Glu or further amino acids that are frequently associated with unstructured regions in natural proteins¹⁸⁸. Non- limiting examples of suitable linker sequences include (GS)5 (GSGSGSGSGS; SEQ ID NO: 23), (GS)10 (GSGSGSGSGSGSGSGSGSGS; SEQ ID NO: 24), (G4S)3 (GGGGSGGGGSGGGGS; SEQ ID NO: 25), llama lgG2 hinge (AHHSEDPSSKAPKAPMA; SEQ ID NO: 26) and human IgA hinge (SPSTPPTPSPSTPPAS; SEQ ID NO: 27). For certain applications, it may be advantageous that the linker molecule comprises or consists of one or more particular sequence motifs. For example, a proteolytic cleavage site can be introduced into the linker molecule such that detectable label or moiety can be released. Useful cleavage sites are known in the art, and include a protease cleavage site such as Factor Xa cleavage site having the sequence IEGR (SEQ ID NO: 28), the thrombin cleavage site having the sequence LVPR (SEQ ID NO: 29), the enterokinase cleaving site having the sequence DDDDK (SEQ ID NO: 30), or the PreScission protease cleavage site having the sequence LEVLFQGP (SEQ ID NO: 31 ).

Alternatively, in case the binding agent is linked to a detectable label or moiety using chemoenzymatic methods for protein modification, the linker moiety may exist of different chemical entities, depending on the enzymes or the synthetic chemistry that is used to produce the covalently coupled molecule in vivo or in vitro'⁶⁹.

A further aspect of the invention relates to a pharmaceutical composition comprising the allosteric modulator as described herein. In particular, a pharmaceutical composition is a pharmaceutically acceptable composition; such compositions are in various embodiments further comprising a (pharmaceutically) suitable or acceptable carrier, diluent, and/or stabilizer, as described herein.

It should be noted that while specific embodiments, configurations, materials, and/or molecules have been discussed herein for methods and products according to the disclosure, various changes or modifications in form and detail may be implemented without departing from the scope of this invention. The following examples are provided to better illustrate particular embodiments, and they should not be construed as limiting the application. The scope of the application is limited solely by the claims.

Table 1 . Amino acid sequences of polypeptides referenced in this disclosure.

Ill

Table 2. Amino acid names and codes used throughout this disclosure.

Aspects of the disclosure A first aspect of the disclosure relates to an allosteric modulator of a protein complex, wherein the protein complex comprises at least one immune receptor and at least one ligand of said immune receptor, and wherein the allosteric modulator specifically binds to a conformation unique to said protein complex, and wherein said modulator, upon binding, induces positive cooperativity to the protein complex.

More specifically, the allosteric modulator of said protein complex, wherein said complex comprises at least one inhibitory immune receptor a nd at least one ligand of said inhibitory immune receptor.

Even more specifically, the disclosure relates to said allosteric modulator, wherein the protein complex comprises at least one immune receptor set forth in Figure 1 and at least one ligand of said immune receptor set forth in Figure 2. Furthermore, wherein the immune receptor of Figure 1 and the ligand of Figure 2 are provided as protein-protein interactors in Figure 3.

Further described is said allosteric modulator, wherein said positive cooperativity induction is determined by a factor a value higher than 1 .

And more preferably, said allosteric modulator, wherein the modulator, upon binding to said protein complex, modulates downstream signaling, most preferably wherein the modulator has spatiotemporal selectivity for the complex.

Also described herein is said allosteric modulator, wherein said allosteric modulator is unique to the conformation of said protein complex by binding to an epitope that is at least partially located on one immune receptor and at least partially located on one ligand engaged in said protein complex.

More specifically, the disclosure relates to said allosteric modulator, wherein said allosteric modulator is an antibody, an active antibody fragment, an immunoglobulin single variable domain (ISVD), a single domain antibody, a VHH, or a nanobody (Nb).

A further aspect relates to said allosteric modulator for use in the treatment of an autoimmune disease.

A final aspect of the disclosure concerns a method for producing said allosteric modulator of a protein complex, the method comprising the steps of: a. Selecting, from a collection of candidate modulators, a modulator or a plurality of modulators that specifically bind to a conformation unique to said protein complex, and b. Identifying, among the selected modulator(s) in a), the allosteric modulator(s) of said protein complexthat induce positive cooperativity to the protein complex upon bindingto it, c. And optionally, identifying, among the selected modulator(s), allosteric modulator(s) of said protein complex that are capable of modulating downstream signaling.

More specifically, said method, wherein the positive cooperativity is determined by a factor a value higher than 1 .

Even more specifically, said method, wherein said allosteric modulator is unique to the conformation of said protein complex by binding to an epitope that is at least partially located on one immune receptor and at least partially located on one ligand engaged in said protein complex.

Further the disclosure specifies said method, wherein said collection of candidate modulators is a library comprising: antibodies, active antibody fragments, immunoglobulin single variable domains (ISVDs), single domain antibodies, VHHs, or nanobodies (Nbs).

More preferably, said method is disclosed, wherein said antibodies, active antibody fragments, ISVDs, single domain antibodies, VHHs, or Nbs are obtained from immunization of a non-human animal with said protein complex in a cross-linked form, preferably wherein the non-human animal belongs to the biological family Camelidae.

Even more preferably, said method is disclosed, wherein said selection of modulator(s) from a collection of candidate modulators in step a) involves displaying candidate modulators on a cell surface via display biopanning, including phage display, yeast display, or mammalian display.

EXAMPLES

Example 1 . The cubic ternary complex model of allosteric modulation of receptor activation.

Most receptor superfamilies are characterized by spatially distinct yet conformationally interconnected binding sites. Allosteric interactions between these topographically separate sites are facilitated through reversible conformational changes, commonly referred to as "allosteric transitions". This mechanistic principle is exemplified by the widely accepted "cubic ternary complex model"²⁹’¹⁹⁰, which describes the dynamic allosteric modulation of receptor activation (Figure 4). This model postulates that a receptor can adopt an inactive (R) or active (R*) state, coupling these allosteric transitions to the biological activity of the receptor. The (R) and (R*) states can be selectively targeted by (natural/endogenous) ligands (L) bindingto the receptor's orthosteric site, and by modulators, such as conformational/allosteric Nanobodies (Nb), that bind to the receptor's allosteric sites. All these interactions may be quantified by unconditional isomerization (K act) and dissociation constants (KL, Kub).

The model includes four key cooperativity factors: (i) a, the factor of binding cooperativity between the orthosteric agonist L and the allosteric modulator Nb; (ii) p, the factor of cooperativity between the orthosteric agonist binding (L) and receptor activation (R -> R*); (iii) y, the factor of cooperativity between the allosteric modulator binding (Nb) and receptor activation (R -> R*); and (iv) 6, the factor of cooperativity between the allosteric modulator (Nb) and agonist (L)-induced receptor activation. These factors yield conditional isomerization and dissociation constants, reflecting the dynamic interplay between ligand binding and receptor conformational states. Specifically, the effective affinity of the orthosteric ligand L for the receptor is modulated by the presence of the allosteric modulator (Nb), and vice versa, with this reciprocal modulation governed by the cooperativity factor a. The probability of receptor activation (R -> R*) in the presence of L and/or Nb is influenced by p, y, and 6, collectively determining how binding events shift the equilibrium between inactive and active states under various conditions. Therefore, the isomerization and dissociation constants serve as quantitative measures of the allosteric linkages that govern the transitions between various receptor conformations, thereby linking receptor structure to its functional state.

The model presented in this Example encapsulates the principles underlying allosteric receptor modulation and offers a quantitative framework for distinguishing between different classes of allosteric ligands. Specifically, it differentiates "allosteric agonists", which are capable of activating the receptor in the absence of an orthosteric agonist, from "allosteric modulators", which do not independently activate the receptor but instead modulate its response to an orthosteric ligand - either enhancing (positive modulation) or diminishing (negative modulation) its effect.

Example 2. Generation of allosteric Nanobodies that stabilize immune checkpoint complexes and function as allosteric modulators.

Allosteric conformation-specific polypeptides, such as antibodies or Nanobodies, may be raised against immunoinhibitory receptor-ligand complexes rather than against the unbound subunits/protomers. Consequently, they can selectively bind and stabilize complexes that contain the active state receptor (R*, see Example 1 ), enhancing immune receptor efficacy through allosteric modulation. In this invention, given the generally transient nature of protein-protein interactions, immune receptor-ligand complexes were stabilized using (bio)chemical cross-linking methods. Next, llamas were immunized with these cross-linked complexes, which closely mimic native protein assemblies in covalent associations. This process elicited an immune response in the llamas, resulting in the production of Nanobodies specifically targeting allosteric epitopes exposed on the surface of the stabilized immune receptor-ligand complexes (Figure 5A).

Using this approach, we generated allosteric binders (i.e., allosteric Nanobodies) directed to conformational epitopes of the cross-linked hPD-1 »hPD-L1 complex. These allosteric binders can induce various effects on the hPD-1 receptor, including: (i) modulation of hPD-1 affinity for its orthosteric ligand hPD-L1 , and/ (ii) modulation of hPD-1 activity (Figure 5B).

Example 3. Immunization of a llama with a cross-linked hPD-1 •hPD-L1 complex to generate an antibody immune library.

The full-length sequences of human PD-1 (SEQ ID NO: 08; PDCD1 , UniProt ID: Q15116) and human PD-L1 (SEQ ID NO: 33; CD274, UniProt ID: Q9NZQ7), as well as their respective extracellular domains, i.e. residues 33-150 of hPD-1 (SEQ ID NO: 06) and residues 18-134 of hPD-L1 (SEQ ID NO: 07), are provided herein. For recombinant expression, only the gene segments encoding the extracellular domains (that is, of SEQ ID NO: 06 or SEQ ID NO: 07) were cloned into a pET-15b expression vector. His-tagged hPD-1 and hPD-L1 proteins were expressed separately in E. coli BL21 (DE3) in inclusion bodies and subsequently refolded according to Zak etal., 2016'⁹ Protein refolding, integrity, purity and functionality were evaluated by size exclusion chromatography, SDS- PAGE and biolayer interferometry (BLI) on Octet (data not shown).

To covalently stabilize the hPD-1 »hPD-L1 complex, glutaraldehyde was used as a chemical crosslinker in the concentration range from 0.29% to 0.04% (Figure 6). 6 pg of hPD-1 , hPD-L1 or the hPD- 1 »hPD-L1 complex was incubated with the respective concentrations of glutaraldehyde at 20°C in a total volume of 15 pl. The reaction was stopped after 45 minutes by the addition of 10 mM Tris- HCl, pH 7.4. The level of cross-linking was monitored by SDS-PAGE and western blot. As illustrated in Figure 6, the addition of glutaraldehyde causes the formation of a higher molecular weight band corresponding to the cross-linked hPD-1 »hPD-L1 complex. A western blot analysis using an anti- hPD-1 antibody (Origene, OTI6F5) detected this band only when both hPD-1 and hPD-L1 were incubated together with glutaraldehyde.

To prepare a crosslinked hPD-1 »hPD-L1 complex for immunization, a mixture of hPD-1 and hPD-L1 (700 pg each) was incubated with 0.05 % glutaraldehyde at 20°C in a total volume of 1700 pL. The reaction was stopped after 45 minutes by the addition of 10 mM Tris-HCl, pH 7.4. The level of crosslinking was monitored by SDS-PAGE and western blot analysis.

A llama was immunized with cross-linked hPD-1 »hPD-L1 complex once a week for six weeks, receiving a total of 740 pg of cross-linked hPD-1 »hPD-L1 complex. After immunization, a blood sample was collected to clone a diverse set of affinity matured Nanobodies with specificity for the hPD-1 »hPD-L1 assembly. Peripheral blood lymphocytes (PBLs) were isolated from the noncoagulated blood for total RNA isolation and subsequent cDNA synthesis. This cDNA served as a template to amplify the open reading frames coding for the variable domains (Nanobodies) of the heavy-chain antibodies; Nanobody fragments were then cloned into an appropriate phage display vector¹⁹².

Example 4. Selection of allosteric Nanobodies that bind and stabilize the hPD-1 »hPD-L1 complex.

The approach employed here to discover Nanobodies that bind to and stabilize the hPD-1 »hPD-L1 complex involves using llama immune libraries and combining phage display with multiple successive biopanning steps, as illustrated in Figure 7.

In a first round (Round 1 a) we immobilized 1 mg of biotinylated hPD-1 (hPD-1 -hFc-Avi-His, AcroBiosystems, PD1 -H82F4) on a neutravidin-coated ELISA plate in the presence of 0.4 % of skimmed milk in PBS. Forthe first round of panning, the immune library was mixed with an excess (1 mM) of soluble hPD-L1 , and next added to the immobilized hPD-1 and incubated for 2 hours at 20°C. Following, several washing steps were performed with PBS before recovering the bound phage after trypsin digestion¹⁹². Eluted phages were next amplified by infecting a freshly grown E. coli culture to recover part of the initial repertoire enriched in antibodies (Nanobodies) that bind to hPD-1 alone or to the hPD-1 »hPD-L1 heterodimer. Because there was an excess of soluble hPD-L1 , phages that bound preferentially to unbound hPD-L1 were counter-selected in Round 1 a.

Phages enriched in Round 1 a were then used as the input for selection in Round 1 b, where the positions of hPD-1 and hPD-L1 were reversed, with hPD-L1 immobilized and hPD-1 in solution, repeating the process. 1 mg of biotinylated hPD-L1 (hPD-L1-hFc-Avi-His, AcroBiosystems, PDL- H82F2) was now immobilized on a neutravidin-coated ELISA plate in the presence of 0.4% skimmed milk in PBS. Phages enriched in Round 1a were mixed with an excess (1 mM) of soluble hPD-1 , and next added to the immobilized hPD-L1 and incubated for 2 hours at 20°C. Then several washing steps were performed with PBS before recovering the bound phage after trypsin digestion. Eluted phages were next amplified by infecting a freshly grown E. coli culture to recover part of the Round 1 b repertoire, further enriched in Nanobodies that bind to hPD-L1 alone or to the hPD-1 »hPD-L1 heterodimer. Because of the excess of soluble hPD-1 , phages that preferentially bound to hPD-1 as a protomer were counter-selected in Round 1 b.

Phages enriched after 2 consecutive rounds of biopanning (1 a and 1 b) were then used as the input for 2 additional rounds. In Round 2a, we used immobilized hPD-1 in combination with soluble hPD- L1 . This was followed by a final round of panning (Round 2b), where we used immobilized hPD-L1 in combination with soluble hPD-1 .

This 4-step procedure includes 4 rounds of biopanning that enrich for Nanobodies that specifically bind to the hPD-1 »hPD-L1 heterodimer, as opposed to 2 rounds that counter-select for binders that are selective for the hPD-L1 protomer (1a, 2a) and 2 rounds that counter-select for binders that are selective for the hPD-1 protomer (1 b, 2b), respectively. Thus, each round progressively enriches for Nbs that bind allosteric epitopes on the complex while simultaneously rejecting Nbs that bind to the soluble protomer in excess.

Example 5. Screening for Nbs binding to hPD-1, hPD-L1 or the cross-linked hPD-1»hPD-L1 complex by ELISA.

To select for Nbs that bind to and stabilize the hPD-1 »hPD-L1 complex, we conducted 4 consecutive rounds of biopanning. In each round, we alternated between immobilizing hPD-1 and hPD-L1 as the protomer while supplementingthe other protomer in solution (see Example 4). This 4-round procedure yielded clones, each containing a phagemid that allows to display the encoded Nanobody on a phage (when using a helper phage) or to express the encoded Nanobody in the periplasm of E.col ⁹². To screen for binders to hPD-1 , hPD-L1 or the complex thereof, we grew individual clones in a 96-well format, induced them with isopropyl 0-D-1 -thiogalactopyranoside (IPTG) and prepared periplasmic extracts as described previously¹⁹². Nanobodies in periplasmatic extracts were tested for binding to hPD-1 alone, hPD-L1 alone or to the cross-linked complex of hPD-1 »hPD-L1 using a standard ELISA protocol. To this end, 0.1 pg of hPD-1 -hFc (AcroBiosystems, PD1 -H5257), hPD-L1-hFc (AcroBiosystems, PD1 -H5258) or the cross-linked complex of hPD- 1 »hPD-L1 were immobilized on Nunc-immuno Maxisorp ELISA plates (Thermo Scientific, 439454), while non-coated wells served as a control. A periplasmic extract lacking VHH expression was used as a negative control. Binding was detected using the CaptureSelect™ Biotin Anti-C-tag Conjugate (Thermo Scientific, 7103252500), which specifically recognizes the C-terminal EPEA-tag on each nanobody (Nb), followed by detection with a streptavidin-alkaline phosphatase conjugate (Thermo Fisher, 7103252100) (Figure 8). Clones identified as binders were subsequently subjected to sequence analysis.

Based on the ELISA screening assay, a set of nanobodies was identified that bind specifically to the crosslinked hPD-1 »hPD-L1 complex but not to the individual protomers. Sequence analysis revealed that these include clones expressed in wells A4, A9, B4, C2, C4, C11 , D4, D5, F9, F10, G1 , G2, E2, E9, and E11 , all of which are sequence variants of CA18811 (SEQ ID NO: 12), as well as clones from wells C7 and F11 , which are sequence variants of CA19275 (SEQ ID NO: 13). Additionally, nanobodies that bind both the hPD-1 »hPD-L1 complex and hPD-L1 alone were identified. These include clones from wells A8, A10, A12, B6, B7, B10, B11 , C3, C5, C9, D3, E7, E8, F2, F3, G4, G5, G6, and G11 , all of which were found to be sequence variants of CA19281 (SEQ ID NO: 15).

Example 6. Screening for Nanobodies that stabilize the non-cross-linked hPD-1*hPD-L1 complex by ELISA.

To identify a potential allosteric modulator of the interaction between hPD-1 and its orthosteric ligand hPD-L1 , we developed an ELISA protocol to assess which Nanobodies increase the affinity of the ligand for the receptor. Accordingly, we coated 10 ng of hPD-1 (hPD-1 -hFc, AcroBiosystems, PD1 -H5257) on 96-well ELISA plates and incubated the coated wells with sub-saturating amounts (1 nM) of biotinylated hPD-L1 (hPD-L1 -hFc-Avi, Tebubio, 71105-2) together with periplasmic extracts containing individual Nanobodies. After washing, PD-1 -PD-L1 complex formation was visualized with a streptavidin-alkaline phosphatase conjugate (Streptavidin Alkaline Phosphatase, Promega, V5591). In each plate a control containing a periplasmic extract of an irrelevant Nb was included as reference, thus not interfering with complex formation (well H12). An increase in signal compared to this control well indicated that the Nanobody stabilizes the interaction between hPD- 1 and hPD-L1 . A decrease in signal indicated that the Nanobody inhibits the interaction between hPD-1 and hPD-L1 (Figure 9).

Example 7. Several allosteric Nanobodies bind to the hPD-1»hPD-L1 complex but not to the separate (unbound) protomers.

Building upon the framework established in Example 2, and based on the rationale that conformation-specific antibodies raised against immune checkpoint complexes, rather than individual protomers, are likely to selectively bind and stabilize these complexes, we immunized a llama with a cross-linked hPD-1 »hPD-L1 complex (see Example 3). Next, we subjected an immune library derived from this animal to 4 rounds of biopanning to enrich for Nanobodies that specifically bind to the hPD-1 »hPD-L1 heterodimer. This process included 2 rounds aimed at counter-selecting binders that selectively target the PD-1 protomer and another 2 rounds aimed at counter-selecting binders that selectively target the PD-L1 protomer (Example 4).

Next, we evaluated selected Nanobodies for their binding affinity to the hPD-1 »hPD-L1 complex, hPD-1 alone, or hPD-L1 alone using BLI on Octet-Red (Sartorius). To this end, purified Nbs were produced. Nbs were expressed in the periplasm of E.coli and purified by affinity chromatography on Ni-NTA using the C-terminal His-tag, followed by size exclusion chromatography in PBS. The purity and integrity was confirmed by SDS-PAGE. Nbs were subsequently biotinylated using a fivefold molar excess of EZ-Link™ NHS-PEG4-Biotin (Thermo Scientific, A39259) for 30 min. The reaction was halted by adding Tris-HCl, pH 8.0, followed by extensive dialysis against PBS buffer.

Biotinylated Nbs were loaded on streptavidin-coated biosensors (Sartorius, 18-5021 ). Recombinant hPD-1 and hPD-L1 proteins were purified as described in Example 3. Each binding experiment followed the following steps: a 1 -minute wash in assay buffer (20 mM Hepes, pH 7.4, 100 mM NaCl, 1 % BSA, 0.05 % Tween 20), immobilization of 1 nmol of biotinylated Nb for3 minutes, another 1 -minute wash. The association with hPD-1 , hPD-L1 , or hPD-1 »hPD-L1 in a concentration range from 2 pM to 125 nM (by a 2-fold serial dilution) was measured for 10 minutes, followed by a dissociation step in assay buffer for 16 minutes. Kinetic binding parameters were assessed by determination of the respective association (K_on) and dissociation rates (K_off) after fitting according to the 1 :1 Langmuir binding model¹⁹³ to calculate the K_d (Kd=K_Off/K_on) values.

When using equimolar amounts of the receptor (hPD-1 ) and the ligand (hPD-L1), we identified several Nanobodies (e.g. CA18811 (SEQ ID NO: 12), CA19275 (SEQ ID NO: 13), CA19281 (SEQ ID NO: 15)) that bind reversibly to the hPD-1 »hPD-L1 complex. However, these same Nanobodies exhibit minimal to no binding to the individual protomers (Figure 10). It is important to note that binding to the hPD-1 »hPD-L1 complex constitutes a trimolecular interaction, whereas binding to the individual protomers represents a bimolecular interaction. Consequently, the kinetic parameters (kon, k_off, and K_d) are reported as apparent values, and alternative binding models may be more appropriate for accurate fitting.

Example 8. Validation of Nanobodies that act as allosteric affinity modulators of the hPD- 1 »hPD-L1 complex by BLI on Octet.

Example 7 describes several Nbs that preferentially bind to the hPD-1 »hPD-L1 complex, compared to the separate protomers, i.e., hPD-1 or hPD-L1 . However, comparing the binding of an antibody to a binary complex (hPD-1 »hPD-L1 ) with its binding to the separate protomers (hPD-1 alone or hPD-L1 alone) is challenging because the properties of ternary complexes must be contrasted with those of binary complexes. To address these challenges related to molecularity, we compared the equilibrium dissociation constant (K_d) of hPD-1 for hPD-L1 (without an allosteric Nanobody) with the affinity of hPD-1 for hPD-L1 in the presence of saturating concentrations (5 pM) of specific allosteric Nanobodies. This comparison was used to estimate the cooperativity factor a. Herein, factor a is the measure of binding cooperativity between the orthosteric ligand (hPD-L1 ) and the allosteric Nanobody (see Example 1).

Accordingly, we immobilized the biotinylated ectodomain of the human hPD-L1 of SEQ ID NO: 07 (Example 3) on a streptavidin-coated Octet biosensor, and incubated such sensors with increasing amounts of the hPD-1 ectodomain (SEQ ID NO: 06) to measure the hPD-1 »hPD-L1 complex association and dissociation in the presence or absence of a given Nb (5 pM). The following allosteric Nanobodies were analyzed: CA18808 (SEQ ID NO: 9), CA18809 (SEQ ID NO: 10), CA18810 (SEQ ID NO: 11 ), CA18811 (SEQ ID NO: 12), CA19275 (SEQ ID NO: 13), CA19279 (SEQ ID NO: 14) and CA19281 (SEQ ID NO: 15). Association and dissociation were monitored, and the maximal binding response was calculated in each condition by background subtraction of parallel sensors. Binding saturation curves were obtained by plotting the relative increase in binding level versus the concentration of hPD-1 (Figure 11). The cooperativity factors awere calculated as the ratio of the equilibrium dissociation constant (K_d) of the complex composed of hPD-1 and hPD-L1 divided by the (apparent) K_d of the ternary complex, wherein the ternary complex is saturated with the allosteric Nanobody.

All of the tested nanobodies function as positive allosteric modulators of the hPD-1 »hPD-L1 complex, as evidenced by cooperativity factor (a) values greater than 1 .

Example 9. Nanobodies act as allosteric affinity modulators of the hPD-1 •hPD-L1 complex on Jurkat cells expressing hPD-1 .

Using the recombinant ectodomains of hPD-1 and hPD-L1 in BLI, we validated several Nanobodies as allosteric affinity modulators of the hPD-1 »hPD-L1 complex (Example 8). To further support these data, we also evaluated if such Nanobodies act as affinity modulators of hPD-L1 for the hPD- 1 receptor expressed on human cells. For these experiments, Jurkat cells stably transfected to express an engineered human PD-1 (hPD-1 ) on the cell surface (Eurofins, DiscoverX; see Example 12 for details) were utilized. Binding of fluorescently labeled hPD-L1 (hPD-L1-His-PE, BioTimes, P1009H6-25) was assessed by FACS in the presence or absence of allosteric nanobodies.

The various Nanobodies of this invention, along with benchmark PD-1 agonist antibodies peresolimab (MedChemExpress, HY-P9993) and rosnilimab (MedChemExpress, HY-P9997), were serially diluted in three-fold steps starting from a concentration of 100 nM. These were then mixed with a constant concentration of hPD-L1 -his-PE (10 nM). Freshly grown Jurkat cells were diluted in U-bottom 96 wells plates (1x10⁵ cells/well), stored for 15 minutes on ice and incubated with the antibodies or nanobodies at 4°C for 30 min with gentle shaking. The cells were then centrifuged and washed twice with cold PBS buffer. Subsequently, the cells were gated and hPD-L1-PE fluorescence was quantified using a BD-Fortessa LSR. Data were analysed using FlowJo software. Each experiment was performed in triplicates.

These experiments demonstrated that CA18811 (SEQ ID NO: 12), CA19275 (SEQ ID NO: 13), and CA19281 (SEQ ID NO: 15) enhanced the binding of fluorescently labeled hPD-L1 to the hPD-1 receptor expressed on the surface of Jurkat cells in a dose-dependent manner. In contrast, neither an irrelevant Nanobody nor the benchmark PD-1 agonist antibodies peresolimab and rosnilimab affected the binding of hPD-L1 to hPD-1 under the same conditions (Figure 12).

Example 10. X-ray structure of CA19279 in complex with the hPD-1 ectodomain.

CA19279 (SEQ ID NO: 14) is a Nanobody that acts as an affinity modulator of the hPD-1 »hPD-L1 complex (Figure 13A; Example 8). CA19279 was selected based on its ability to bind to a complexspecific conformation, i.e., hPD-1 »hPD-L1 complex-specific conformation. To confirm that this Nanobody binds an allosteric epitope, we solved the structure of this Nanobody in a complex with hPD-1 usingX-ray crystallography (Figure 13B). Purified hPD-1 and CA19279 (SEQ ID NO: 14) were mixed in a 1 :2 molar ratio, and the complex was purified by gel filtration (Cytiva, Superdex Increase 10/300 GL) in a buffer containing 20 mM Hepes, pH 7.4, 50 mM NaCl. Fractions containing the CA19279»hPD-1 were pooled and concentrated to 7 mg/ml. Diffraction-quality crystals were grown using a sitting-drop vapor diffusion setup with a crystallization solution consisting of 0.1 M sodium acetate at pH 5, 2% (w/v) PEG4000, and 15% (v/v) MPD. The crystal comprising hPD-1 (SEQ ID NO: 8) and CA19279 (SEQ ID NO: 14) is in the space group P 1 21 1 , with the following lattice constants: a= 66.506A, b=39.038A, c= 91.475A, a=90°, 0= 101.413°, y=90°. The crystals diffracted at a resolution of 1 .8 A.

The ectodomain of hPD-1 consists of a “front” 0-sheet face comprising the CC'FG strands and a “back” 0-sheet face comprising the AA'BDE strands¹⁹⁴. hPD-L1 binds the “front” 0 sheet of hPD-1 (PDB:4ZQK). In the CA19279»hPD-1 complex, the CDR3 of CA19279 (SEQ ID NO: 19) adopts a 0- strand conformation allowing three of its residues (Asp99, Arg101 , Tyr103) to form hydrogen bonds with Ala40, Leu41 , Val43, Thr145 of hPD-1 . The binding epitope of CA19279 is thus mainly located on the “back” of hPD-1 , including the A’ 0-strand of hPD-1 (residues Ala40 to Val44)¹⁹⁵. All intermolecular hydrogen bonds between CA19279 and hPD-1 are listed in Figure 13C.

When comparing the obtained crystal structure of the CA19279»hPD-1 complex with structures of the hPD-1 »PD-L1 complex (PDB: 4ZQK), it becomes evident that CA19279 binds to a nonoverlapping allosteric epitope located on the "back" 0-sheet face (the A' strand) of hPD-1 , opposite the orthosteric binding site located at the "front" 0-sheet face of hPD-1 , wherein PD-L1 binds.

Example 11. Nanobodies that act as affinity modulators of the hPD-1 •hPD-L1 interaction do not modulate the hPD-1»hPD-L2 complex.

The hPD-1 receptor is triggered by different natural ligands including hPD-L1 and hPD-L2. To determine whether allosteric Nanobodies that act as affinity modulators of the hPD-1 »hPD-L1 interaction are ligand-specific and do not affect the hPD-1 »hPD-L2 interaction, we compared the impact of allosteric modulators CA18811 (SEQ ID NO: 12), CA19275 (SEQ ID NO: 13), CA19281 (SEQ ID NO: 15), and an irrelevant Nb of SEQ ID NO: 16 (targeting an irrelevant protein) on the binding properties of hPD-L1 and hPD-L2 to hPD-1 using ELISA.

Similar to Example 6, 10 ng of hPD-1 (hPD-1 -hFc, AcroBiosystems, PD1 -H5257) was immobilized on plates and incubated with serial three-fold dilutions (starting at 200 ng/mL) of either biotinylated hPD-L1 (hPD-L1-hFc-Avi, Tebubio, 71105-2) or biotinylated hPD-L2 (hPD-L2-hFc-Avi, AcroBiosystems, PD2-H82F6) in the presence of various Nanobodies and control antibodies. Nanobodies CA18811 (SEQ ID NO: 12), CA19275 (SEQ ID NO: 13), and CA19281 (SEQ ID NO: 15) were tested at a concentration of 100 nM. The benchmark PD-1 agonist antibodies peresolimab (MedChemExpress, HY-P9993) and rosnilimab (MedChemExpress, HY-P9997) were used at 10 nM and 0.5 nM, respectively. An irrelevant Nanobody (SEQ ID NO: 16), specific for an unrelated target, was included as a negative control at 100 nM

After washing, plates were developed using a streptavidin-alkaline phosphatase conjugate (Promega, V5591 ). An increase in signal corresponds to stabilization of the ligand-receptor interaction by the tested Nanobody or antibody in a concentration-dependent manner (Figure 14).

As shown, all test Nanobodies - but not peresolimab or rosnilimab - acted as positive affinity modulators of the hPD-1 »hPD-L1 interaction. None of the molecules influenced the hPD-1 »hPD-L2 interaction, indicating that the effects of CA18811 (SEQ ID NO: 12), CA19275 (SEQ ID NO: 13), and CA19281 (SEQ ID NO: 15) on hPD-1 »hPD-L1 complexation are specific to the PD-L1 ligand.

Example 12. Nanobodies that behave as allosteric affinity modulators of the hPD-1 •hPD-LI complex improve the recruitment of SHP1 to hPD-1 in cells in a hPD-L1-dependent manner.

JurkatT cells overexpressing full length hPD-1 fused to an enzyme donor (ED) domain and a hSHP1 domain fused to an enzyme acceptor (EA) domain were purchased from Eurofins DiscoverX. The principle of the assay described here relies on the intracellular recruitment of SHP1 to hPD-1 upon activation, resulting in the complementation of the two enzyme fragments to form active betagalactosidase. Upon using a specific substrate, the beta-galactosidase activity is measured through luminescence, directly correlating with SHP1 recruitment to the activated hPD-1 receptor.

Raji B cells stably expressing hPD-L1 (Raji-hPD-L1) and the corresponding parental control cells lacking PD-L1 expression (Raji-Null) were obtained from InvivoGen. These cells were used to present hPD-L1 to the reporter Jurkat cells and to serve as a control for ligand selectivity, respectively. Both cell lines express the Fc receptor FCR2B (as annotated in the Human Protein Atlas), facilitating optimal engagement by the Fc-containing benchmark antibodies. Briefly, the test compounds were diluted in PBS to 5 times the desired final concentration and added to a white 96-well tissue culture plate (20 pL/well). Next, 40 pL of Raji cells (hPD-L1 or Null) at the desired concentration (5.000, 10.000, or 20.000 cells/well as indicated) were added, followed by 40 pL of Jurkat reporter cells (10.000 cells/well). The mixture was incubated for 1 hour at 37°C. The plates were then equilibrated at room temperature for 1 hour before adding the betagalactosidase substrate and reading luminescence according to the manufacturer’s protocol.

As shown in Figure 15, incubation of Jurkat reporter cells with Raji-Null cells yielded a uniform signal across all tested cell numbers, reflecting background SHP1 recruitment. In contrast, coculture with Raji-hPD-L1 cells produced a dose-dependent increase in signal intensity, consistent with ligand-dependent engagement of hPD-1. Based on these results, a dose of 10,000 Raji cells per well was selected for subsequent experiments. At this cell density, Raji-hPD-L1 cells generated a robust signal window - approximately a sixfold increase relative to the Raji-Null control - while remaining within the linear dynamic range and avoiding saturation of hPD-1 receptor occupancy.

Figure 16 presents data normalized to their respective baselines, defined as the signal from Jurkat reporter cells incubated with either 10,000 Raji-Null or 10,000 Raji-hPD-L1 cells in the presence of PBS only. In Figure 16A, representative PD-1 antagonists were evaluated. The anti-PD-1 antagonist Nanobody 102C12 (disclosed in WO2017/087587), which is known to block the interaction between hPD-1 and hPD-L1 , inhibited the engagement of Raji-hPD-L1 cells with the reporter Jurkat cells. This inhibition resulted in a dose-dependent reduction in signal relative to the Raji-hPD-L1 baseline. In contrast, in the absence of hPD-L1 (i.e. , when Raji-Null cells were used), the reporter signal remained at baseline across all tested doses of 102C12. Pembrolizumab, a clinically approved anti-PD-1 antagonist antibody¹⁹⁶, similarly reduced the reporter signal induced by Raji- hPD-L1 cells in a dose-dependent manner. Interestingly, in the absence of hPD-L1 (i.e., with Raji- Null cells), pembrolizumab increased the reporter signal. This unexpected effect may be attributed to the antibody’s capacity to crosslink and cluster PD-1 molecules on the Jurkat cell surface, potentially leading to receptor activation.

Regarding the PD-1 agonist antibody activity shown in Figure 16B, rosnilimab¹⁹⁷ dose-dependently increased the signal of SHP1 recruitment in the presence of both Raji-Null and Raji-hPD-L1 , as expected from a ligand-independent agonist. The effect was much stronger in the absence of hPD- L1 (maximum 2-fold signal increase for Raji-hPD-L1 vs. 17-fold for Raji-Null), likely because hPD-1 is already partially activated by the presence of hPD-L1 , reducing the remainingwindowfor further activation. This profile was even more pronounced for the other agonist, peresolimab, whose activity was weaker than that of rosnilimab and could only be detected in the absence of hPD-L1 . In contrast to all these benchmarks, the three hPD1 »hPDL1 stabilizing Nanobodies selected herein present a unique profile (Figure 16C). While the irrelevant Nanobody remained inactive under all conditions, all stabilizing Nanobodies tested were inactive in the absence of hPD-L1 (Raji-Null) but showed a dose-dependent increase in SHP1 recruitment signal in the presence of hPD-L1 positive cells (Raji-hPD-L1). This indicates their ability to potentiate ligand-dependent receptor activation; hence, these stabilizers enhance hPD-1 activation and downstream SHP1 recruitment only in the presence of its cognate ligand, hPD-L1 .

Under these conditions, CA19281 (SEQ ID NO: 15) was the most potent with an EC₅o of 145 nM, followed closely by CA19275 (SEQ ID NO: 13) at 161 nM, and CA18811 (SEQ ID NO: 12), which was slightly less active at 344 nM (Figure 17).

In conclusion, these data indicate that Nanobodies functioning as positive allosteric affinity modulators of the hPD-1 »hPD-L1 complex, such as CA18811 (SEQ ID NO: 12), CA19281 (SEQ ID NO: 15), and CA19275 (SEQ ID NO: 13), enhance the recruitment of SHP1 to hPD-1 in a hPD-L1- dependent manner. Thus, such Nanobodies function as positive allosteric modulators (PAMs) specific for the hPD-1 »hPD-L1 complex, capable of modulating hPD-1 signaling in vitro in a strictly ligand-specific manner.

Example 13. Nanobodies that behave as allosteric affinity modulators of the hPD-1 •hPD-L1 complex inhibit activation of NFAT signaling in JurkatT cells in a hPD-L1 -dependent manner.

The nuclear factor of activated T cells (NFAT) is a key signaling molecule involved in the response of T cells to activation by antigen-presenting cells or by alternative mimicking treatments, such as anti-CD3 antibody treatment. Co-stimulatory (e.g., CD28) and co-inhibitory (e.g., PD-1 ) checkpoints enhance or inhibit NFAT's ability to modify gene expression. Jurkat T cells, which overexpress an active TCR and full-length hPD-1 , and carry a Lucia luciferase reporter under the control of the NFAT promoter (Invivogen), were used to evaluate the ability of hPD-1 pathway modulators to counter TCR activation by assessing their capacity to lower NFAT-induced luminescence upon anti-CD3 treatment.

For this assay, test compounds were diluted in PBS to 5-fold the desired final concentration and added to a 96-well tissue culture plate (20 pL/well). Subsequently, 40 pL of THP-1 cells (10,000 cells/well) along with anti-CD3 antibodies (clone OKT3), and with hPD-L1 -Fc or without, were added. Finally, 40 pL of Jurkat reporter cells (10,000 cells/well) were added and the plate was incubated overnight at 37°C. The final concentrations used were 0.3 pg/mLof anti-CD3 and 3 pg/mL of hPD-L1 -Fc. Followingthe overnight incubation, the plates were equilibrated at room temperature for 1 hour before adding the Lucia luciferase substrate and reading luminescence according to the manufacturer’s instructions.

As depicted in Figure 18A, under baseline conditions (PBS), the addition of hPD-L1 -Fc to the reporter cells decreased anti-CD3-induced NFAT signaling by 1.5-fold, consistent with the expected effect of the inhibitory checkpoint receptor hPD-1 's ligand. In the absence of ligand, none of the tested benchmark molecules affected NFAT signaling relative to their respective controls (lgG1 and mock Nb), with the exception of rosnilimab. As a ligand-independent anti-PD-1 agonist, rosnilimab alone suppressed NFAT signaling. In the presence of hPD-L1 , both the anti-PD-1 antagonist antibody pembrolizumab and the Nanobody 102C12 (as disclosed in WO2017/087587) effectively blocked the inhibitory effect of hPD-L1 on hPD-1 , restoring NFAT signaling to baseline levels observed in the absence of ligand. In contrast, rosnilimab exhibited an additive effect on hPD-L1 -induced suppression of NFAT signaling, resulting in a further decrease in luminescence. However, both hPD-1 »hPD-L1 stabilizing nanobodies, CA18811 (SEQ ID NO: 12) and CA19281 (SEQ ID NO: 15), showed no activity in the absence of ligand but effectively decreased NFAT signaling in a dose-dependent mannerwhen hPD-L1-Fc was present (Figure 18B).

In conclusion, Nanobodies that act as allosteric affinity modulators of the hPD-1 »hPD-L1 complex, such as CA18811 (SEQ ID NO: 12) and CA19281 (SEQ ID NO: 15), counteract TCR-induced NFAT signaling in hPD-1 expressing cells in a PD-L1 -dependent manner.

Example 14. Immunization of a llama with a cross-linked hPD-1 -hPD-L2 complex to generate an antibody immune library.

The recombinant ectodomain of the human PD-L2 (hPD-L2) (SEQ ID NO: 241 ; AcroBiosystems, PD2-H5220) was mixed with the hPD-1 ectodomain (SEQ ID NO: 06) to form the hPD-1 »hPD-L2 complex.

To covalently stabilize the hPD-1 »hPD-L2 complex, glutaraldehyde was utilized as a chemical cross-linker across concentrations ranging from 0.25% to 0.03%. A total of 3 pg of either hPD-1 , hPD-L2, or the hPD-1 »hPD-L2 complex was treated with glutaraldehyde at 20°C in a 20 pL reaction volume. The degree of cross-linking was assessed using SDS-PAGE followed by western blot analysis. Upon addition of glutaraldehyde, a band correspondingto the cross-linked hPD-1 »hPD- L2 complex appeared at the expected molecular weight in the western blot (Figure 19). This was visualized using an anti-His antibody (Bio-Rad, MCA1396, mouse anti-Histidine tag), indicating successful formation of the complex only when both hPD-1 and hPD-L2 were co-incubated for cross-linking. To prepare a crosslinked hPD-1 »hPD-L2 complex for immunization, a mixture of hPD-1 and hPD-L2 protein (700 pg each) was incubated with 0.03 % glutaraldehyde at 20°C in a total volume of 1320 pl. The reaction was stopped after 60 minutes by the addition of 10 mM Tris-HCl, pH 8.0. The level of cross-linking was monitored by SDS-PAGE and western blot.

A llama underwent weekly immunizations over six sessions, receiving a total of 940 pg of crosslinked hPD-1 »hPD-L2 complex. Following the immunizations, a blood sample was collected to isolate peripheral blood lymphocytes (PBLs). From these cells, total RNA was extracted and used to synthesize cDNA. The cDNA was subsequentlyutilized as a template to amplify the open reading frames encoding the variable domains (Nanobodies) of the heavy-chain antibodies. These Nanobody fragments were then cloned into a suitable phage display vector¹⁹².

Example 15. Selection of allosteric Nanobodies that bind and stabilize the hPD-1 •hPD-L2 complex.

To induce the immune response for generating allosteric Nanobodies that bind to and stabilize the hPD-1 »hPD-L2 complex, a llama was immunized using a cross-linked complex of hPD-1 »hPD-L2 recombinant proteins. Subsequently, a Nanobody immune library displayed on phage was constructed, following the methodology outlined in Example 14.

For the selection of allosteric Nanobodies (Nbs) that bind to and stabilize the hPD-1 »hPD-L2 complex, 4 rounds of biopanning were performed. In a first round (Round 1 a) we immobilized 1 pg of biotinylated hPD-1 (hPD-1 -hFc-Avi-His, Acrobiosystems, PD1 -H82F4) on a neutravidin-coated ELISA plate in the presence of 0.4% of skimmed milk in PBS. For the first round of panning, the immune library was mixed with an excess (1 pM) of soluble hPD-L2 (hPD-L2-His, Acrobiosystems, PD2-H5220), and next added to the immobilized hPD-1 and incubated for 2 hours at 20°C. Then several washing steps were performed with PBS before recovering the bound phage after trypsin digestion¹⁹². Eluted phages were next amplified by infecting a freshly grown E. cof/cultureto recover part of the initial repertoire enriched in antibodies (Nanobodies) that bind to hPD-1 alone or to the hPD-1 »hPD-L2 heterodimer. Because of the excess of soluble hPD-L2, phage displaying Nanobodies preferentially bindingto hPD-L2 as a monomerwere counter-selected in Round 1 a.

Phages enriched in Round 1 a were then used as the input for selection in Round 1 b, following a reversed protocol where hPD-L2 was immobilized and hPD-1 was soluble. Specifically, 1 pg of biotinylated hPD-L2 (hPD-L2-hFc-Avi-His, Acrobiosystems, PD2-H82F6) was immobilized on a neutravidin-coated ELISA plate in PBS containing 0.4% skimmed milk. The enriched phages from Round 1 a were mixed with excess soluble hPD-1 (1 pM) and added to the immobilized hPD-L2, incubating for 2 hours at 20°C. After several washes with PBS, bound phages were recovered by trypsin digestion. Eluted phages were then amplified by infecting a fresh E. coli culture to recover part of the Round 1 b repertoire, further enriched in Nanobodies that bind to the hPD-1 »hPD-L2 heterodimer. Counter-selection against phages preferentially binding to hPD-1 as a monomer was implemented due to the excess of soluble hPD-1 during Round 1 b.

Phages enriched after 2 consecutive rounds of biopanning (1a followed by 1 b) were next used as the input for 2 more equivalent rounds where we first used immobilized hPD-1 in combination with soluble hPD-L2 (Round 2a) followed by a last round of panning where we used immobilized hPD-L2 in combination with soluble hPD-1 (Round 2b).

The 4-step procedure involves four rounds of biopanning aimed at enriching for Nanobodies specific to the hPD-1 »hPD-L2 heterodimer. Particularly, it includes two rounds (1a, 2a) designed to counter-select for Nanobodies selective for the hPD-L2 monomer and two rounds (1 b, 2b) to counter-select for Nanobodies selective for the hPD-1 monomer. These counter-selection rounds are important for eliminating Nanobodies binding to the soluble monomers that are present in excess. Overall, each round cumulatively enriches for Nanobodies binding to allosteric epitopes on the hPD-1 »hPD-L2 complex while counter-selecting for those binding to the individual monomers.

Example 16. Screening for Nanobodies that stabilize the non-cross-linked hPD-1*hPD-L2 complex by ELISA.

To measure the effects of a potential allosteric modulator on the binding between hPD-1 and its alternative orthosteric ligand hPD-L2, we developed an ELISA setup to measure which Nanobodies increase the affinity of the ligand for the receptor. Accordingly, we coated 10 ng of hPD-1 (hPD-1 - hFc, Acrobiosystems, PD1-H5257) on ELISA plates and incubated coated wells with sub-saturating amounts (0.5 nM) of biotinylated hPD-L2 (hPD-L2-hFc-Avi, AcroBiosystems, PD2-H82F6) together with periplasmic extracts containing individual Nanobodies. After washing, plates were developed with a streptavidin-alkaline phosphatase conjugate (Streptavidin Alkaline Phosphatase, Promega, V5591). Wells H11 and H12 of the ELISA plate served as controls: well H11 contained hPD-L2 in the absence of a Nanobody, while well H12 contained a periplasmic extract expressing an irrelevant Nanobody (SEQ ID NO: 16). An increase in signal compared to these references indicated that the Nanobody stabilizes the interaction between hPD-1 and hPD-L2. Conversely, a decrease in signal indicated that the Nanobody inhibits the interaction between hPD-1 and hPD-L2 (Figure 20). Sequencing revealed that wells B2 and H2 contained sequence variants of CA19998 (SEQ ID NO: 242)

Example 17. Validation of Nanobodies that act as allosteric affinity modulators of the hPD- 1 *hPD-L2 complex by BLI on Octet. Example 15 describes the discovery of Nbs that referentially bind to the hPD-1 »hPD-L2 complex, compared to the separate protomers hPD-1 or hPD-L2. However, comparing the binding of an antibody to a binary complex (hPD-1 »hPD-L2) with its binding to the separate protomers (hPD-1 alone or hPD-L2 alone) is challenging because the properties of ternary complexes must be contrasted with those of binary complexes. To address these challenges, we compared the (bimolecular) equilibrium dissociation constant (K_d) of hPD-1 for hPD-L2 (in the absence of an allosteric Nanobody) with the affinity of hPD-1 for hPD-L2 in the presence of saturating concentrations (500 nM) of Nanobody CA19998 (SEQ ID NO: 242) to estimate the cooperativity factor a. Herein, factor a is the measure of binding cooperativity between the orthosteric ligand (PD-L2) and the allosteric Nanobody (see Example 1).

Accordingly, we immobilized the biotinylated ectodomain of hPD-1 (hPD-1 -hFc-Avi-His, AcroBiosystems, PD1 -H82F4) on a streptavidin-coated Octet sensor and incubated these sensors with increasing concentrations of the hPD-L2 ectodomain (PD-L2-His, AcroBiosystems, PD2- H5220) to assess the kinetic association and dissociation constants in the presence or absence of 500 of CA19998 (SEQ ID NO: 242). Binding saturation curves were derived by plotting the relative increase in amplitude of the signal against the concentration of hPD-L2 (Figure 21). The a cooperativity factor was calculated as the ratio of the equilibrium dissociation constant (K_d) of the binary complex (hPD-1 »hPD-L2) to the apparent K_d of the ternary complex (hPD-1 »hPD- L2»CA19998), wherein the ternary complex is saturated with the allosteric Nanobody. This analysis revealed that CA19998 (SEQ ID NO: 242) enhances the affinity of hPD-1 for hPD-L2 by 17300-fold, underscoring significant cooperativity between this allosteric Nanobody and the orthosteric ligand (hPD-L2).

Example 18. Nanobodies that behave as allosteric affinity modulators of the hPD-1*hPD-L1 or hPD-1»hPD-L2 complexes improve recruitment of SHP1 to PD-1 in cells in a ligand-specific manner.

As in Example 12, Jurkat T cells overexpressing full-length hPD-1 fused to an enzyme donor (ED) domain and a hSHP1 domain fused to an enzyme acceptor (EA) domain were used with Raji B cells not expressing hPD-L1 (Raji-Null) to test the ability of different PD-1 pathway modulators to activate PD-1. In this setup, the ligand for hPD-1 was supplied as soluble hPD-L1 -Fc or hPD-L2-Fc at a concentration of 5 pg/mL. White 96-well assay plates containing the cells, ligands, and the test compounds were incubated for 1 hour at 37°C. The plates were then equilibrated at room temperature for 1 hour before adding the beta-galactosidase substrate and reading luminescence according to the manufacturer’s directions. As shown in Figure 22, in the absence of ligand, all tested molecules remained at baseline, with the exception of rosnilimab (used as a positive control), which markedly increased SHP1 recruitment. Rosnilimab also enhanced SHP1 recruitment in the presence of both hPD-L1 and hPD-L2, consistent with its ability to activate hPD-1 in a ligand-independent manner. In contrast, the anti- PD-1 antagonist Nanobody 102C12 inhibited ligand binding, thereby suppressingSHPI recruitment to baseline levels observed in the absence of ligand, for both hPD-L1 and hPD-L2. In contrast, the anti-PDL2 antagonist nanobody CA19999 was only able to prevent SHP1 recruitment induced by hPD-L2, and not by hPD-L1 .

The hPD-1 »hPD-L2 stabilizer CA19998 (SEQ ID NO: 242) only increased SHP1 recruitment in the presence of hPD-L2, and not hPD-L1 , while the hPD-1 »hPD-L1 stabilizer Nanobody CA19275 (SEQ ID NO: 13) was inactive in the presence of hPD-L2 and acted as an agonist of SHP1 recruitment with hPD-L1. These data demonstrate that positive allosteric modulators (PAMs) specific for hPD- 1 »hPD-L1 or hPD-1 »hPD-L2 complexes were identified herein that modulate hPD-1 signaling/n vitro in a strictly ligand-dependent and ligand-specific manner.

Example 19. Nanobody CA19998 that acts as affinity modulator of the hPD-1*hPD-L2 interaction does not modulate the hPD-1»hPD-L1 complex.

The hPD-1 receptor is activated by different natural ligands, namely hPD-L1 and hPD-L2. To test if allosteric Nanobodies that act as affinity modulators of the hPD-1 »hPD-L2 interaction are ligand specific and do not modulate the hPD-1 »hPD-L1 interaction, the effect of an allosteric affinity modulator of the hPD-1 »hPD-L2 interaction (CA19998 (SEQ ID NO: 242) on the binding kinetics of hPD-L1 or hPD-L2 to hPD-1 was compared with that of an affinity modulator of the hPD-1 »hPD-L1 complex (CA19275; SEQ ID NO: 13), using ELISA.

Similar to Example 11 , ELISA plates were coated with 10 ng of hPD-1 (hPD-1 -hFc, Acrobiosystems, PD1 -H5257) and incubated with biotinylated hPD-L1 (hPD-L1 -hFc-Avi, Tebubio, 71105-2) or biotinylated hPD-L2 (hPD-L2-hFc-Avi, Acrobiosystems, PD2-H82F6) in serial three-fold dilutions starting from 25 ng/ml. This was done in the presence of nanobody CA19998 (SEQ ID NO: 242), CA19275 (SEQ ID NO: 13), or an irrelevant Nb (SEQ ID NO: 16) at concentrations of 100 nM. After washing, streptavidin-alkaline phosphatase conjugate (Promega, V5591 ) was used for detection. In contrast to CA19275 (SEQ ID NO: 13), CA19998 (SEQ ID NO: 242) improved the binding of hPD- L2 to hPD-1 (Figure 23, left panel). When using hPD-L1 as the ligand (Figure 23, right panel), the inverse was observed: CA19275 increased the binding of hPD-L1 to hPD-1 , while CA19998 did not modulate this interaction. Thus, Nanobodies that act as affinity modulators of the hPD-1 »hPD-L1 interaction do not modulate the hPD-1 »hPD-L2 interaction, and vice versa. Example 20. Immunization of a second llama with a cross-linked hPD-1»hPD-L1 complex composed of proteins produced in mammalian cells yielded a new set of Nanobodies that function as allosteric affinity modulators of the complex.

Following the protocol described in Example 3, we produced a cross-linked hPD-1 »hPD-L1 complex (Figure 26) using proteins expressed in mammalian cells (Aero Biosystems, PD1 -H5221 and PD1-H5229), and therefore containing all post-translational modifications (PTMs) expected in the endogenous proteins. This complex was used for a new round of immunization in a second llama (see Example 3 forthe protocol). The resulting immune library was subjected to selection for stabilizers of the hPD-1 »hPD-L1 complex using the panning procedure described in Example 4. Following primary screening by ELISA (method described in Example 5), three representative candidate stabilizing Nanobodies, namely A00111 (SEQ ID NO: 34), A00118 (SEQ ID NO: 41 ), and A00123 (SEQ ID NO: 46), were validated as allosteric affinity modulators with measurable a factors greater than 1 (Table 3 and Figure 27). The a factors were determined by BLI using a setup similar to that described in Example 8, with the exception that for A00111 and A00123, an inverted configuration was employed, i.e., immobilizing hPD-1 on the sensor and titrating hPD-L1 in solution. Furthermore, these new molecules and their related variants were found to recruit SHP1 in hPD-1-expressing cells, but only in the presence of hPD-L1-expressing cells (Table 3; assay described in Example 12).

Table 3. Activity of the library of PAMs generated in this Example.

Example 21. Humanization and optimization of PAM sequences.

Sequences of allosteric affinity modulators can be modified to improve their drug-like properties, as described herewith, for instance by humanization towards human IGHV3 and JH germline consensus sequences, while retaining their functionality and removal of residues problematic for chemical and biophysical stability.

In this context, different mutant variants were generated for multiple PAMs of this invention, and then compared to their respective parental sequences in functional assays (e.g., SHP1 recruitment assays as described in Example 12), and/or biophysical assays (e.g., calculation of the factor alpha, as described in Example 8). For example, optimization was carried out for the identified hPD- 1 »hPD-L1 PAM CA19281 (SEQ ID NO: 15). A first round of mutations was introduced into residues not involved in antigen binding, based on framework alignment of the parental sequence with human IGHV3 and JH germline genes (Figure 28) and on prior knowledge of motifs potentially leading to post-translational modifications¹²³, resulting in a framework-optimized sequence of A00032 (SEQ ID NO: 49). In Example 8, CA19281 was shown to improve the K_d ^app of PD-L1 binding to PD-1 by 2055-fold. In a side-by-side comparison, A00032 demonstrated a 6.7-fold improvement of the hPD-1 »hPD-L1 binding constant in comparison to CA19281 (K_d ^app of 8.1x1 O'⁹ M versus 5.4x10' ⁸ M respectively). Moreover, the a factor for A00032 was calculated (using methods described herewith) to be 1860, further indicating that framework optimization retains the molecule’s allosteric affinity-modulating properties. In agreement with these findings, CA19281 and A00032 had an overlapping potency and efficacy in the SHP1 recruitment assay (Figure 29A), confirming the retained pharmacological activity of the framework optimized variant.

Single mutations toward the human sequence can then be introduced in regions outside the antigen-binding sites that are more likely to influence binding. In the case of A00032, methionine 85 (M85), susceptible to oxidation and located near a hallmark residue, was substituted with threonine (T), resulting in Nanobody A00044, (SEQ ID NO: 50). Alternatively, threonine 68 (T68), located near CDR2, was substituted with alanine (A) to increase similarity to the human sequence, resulting in Nanobody A00043 (SEQ ID NO: 51 ). As seen in Figure 29B, these two single mutations (in A00043 or A00044) had no negative effect on activity in the SHP1 recruitment assay and were thus permitted.

Next, to identify suitable alternatives for residues within the antigen-binding sites (CDRs), mutant libraries were generated for each potentially problematic residue. In the case of A00032, an NS motif, susceptible to deamination, was found in the CDR1 , and methionine, susceptible to oxidation, was present in the CDR3. From the mutant libraries, the N36Q and N36R mutations (for the former) and M109L, M109S, M109D, and M109R mutations (for the latter) preserved modulator functionality in the SHP1 recruitment assay (data not shown).

As a final step, all permitted mutations were combined into a single sequence. For example, the mutations N36Q, T68A, M85T, and M109L yielded the optimized version of A00032, designated nanobody A00235 (SEQ ID NO: 53). As shown in Figure 29C, in the SHP1 recruitment assay, fully sequence-optimized CA19281 variants - carrying stabilizing and humanizing mutations in the framework, as well as at positions N36, T68, M85, and M 109 - exhibited no loss of functionality.

As a second example, the same work of sequence optimization and humanization was carried out on the hPD-1 »hPD-L1 PAM CA18810 (SEQ ID NO: 11). In this case too, the framework optimized version of this parent (A00255, SEQ ID NO: 54) and the further sequence-optimized variant (A00343, SEQ ID NO: 55, which contains 4 additional mutations at residues possibly impacting binding) were similarly potent at recruiting SHP1 (Figure 29D). The activity of selected optimized variants generated in this Example is shown in Table 4.

Therefore, the current Example shows that sequence optimization of the PAM sequences, for instance as disclosed herein, can be performed without compromising their allosteric affinitymodulating properties and pharmacological activity.

The sequences of various optimized nanobody variants generated in this Example encompass SEQ ID NOs: 49, 50, 51 , 53, 54, 55, 56, 57, 58, or 128-184. Table 4. Activity of selected optimized PAMs of this Example.

Example 22. Formatting of PAM sequences.

In addition to sequence optimization, PAMs of the current invention can be formatted for half-life extension and enhanced drug-like properties, among other properties. For example, the framework-optimized version of hPD-1 »hPD-L1 PAM CA19281 , namely A00032 (SEQ ID NO: 49) described in the previous Example, can be fused to the anti-serum albumin (SA) VHH SA26h5 (WO2019016237) via a (GGGGS)7 (SEQ ID NO: 59) linker, which has been shown to improve pharmacokinetic profiles of nanobodies (WO2012175400)^198-201. This format also allows coupling of multiple repeats of the same Nanobody in bi- (e.g., A00055, SEQ ID NO: 60), tri- (e.g., A00153, SEQ ID NO: 61 ), or tetravalent (e.g., A00278, SEQ ID NO: 62) configurations fused to SA26h5. Additionally, it enables coupling of different Nanobodies, as demonstrated by the framework- optimized variants of CA19281 and CA19275 combined in the formatted Nanobody A00137 (SEQ ID NO: 63).

As shown in Figure 30A, these formatted sequences were tested in the SHP1 recruitment assay (described in Example 12) to evaluate the impact of each modification on potency and efficacy. In this example, all formatting strategies either maintained or improved potency compared to the monovalent format. Moreover, while the initial bivalent Nanobody was linked with a (GGGGS)7 (SEQ ID NO: 59) linker (in A00055, SEQ ID NO: 60), we demonstrated that shorter linkers, namely (GGGGS)1 (SEQ ID NO:64) (in A00144, SEQ ID NO: 65), (GGGGS)2 (SEQ ID NO: 66) (in A00147, SEQ ID NO: 67), and (GGGGS)4 (SEQ ID NO: 68) (in A00150, SEQ ID NO: 69), all exhibited similar pharmacological profiles (Figure 30B), indicating that the modulators' activity is retained across different linker lengths between the subunits.

Anotherwell-established strategy for half-life extension is fusion to an Fc domain²⁰². Herewith, the framework-optimized variant of CA19281 - A00032 (SEQ ID NO: 49) - was fused to either a wild-type lgG1 Fc tail (also referred to herein as ‘IgG-fusion Nanobody) (A00062, SEQ ID NO: 70) or a LALAPG (SEQ ID NO: 71 ) mutated Fc tail (A00065, SEQ ID NO: 72). The LALAPG (SEQ ID NO: 71 ) mutations, introduced in the lower hinge and CH2 domain of mouse lgG2a, are known to attenuate Fc effector functions²⁰³. In the SHP1 recruitment assay, both formats demonstrated improved potency (Figure 30C).

As shown in Figure 30D, despite being formatted as bivalent molecules, both A00055 and A00062 retained ligand-dependent activity and were inactive in the absence of PD-L1 -expressing cells. This contrasts with classical bivalent PD-1 antibody agonists, which activate PD-1 via crosslinking²⁰⁴ (see Example 12).

These findings are consistent with biophysical analyses: in a side-by-side comparison, the binding constant of the PD-1 »PD-L1 complex in the presence of A00032 (K_d ^app = 8.1 * 10“⁹ M, over 2,000- fold higher affinity than without a nanobody; see Example 21 ) was not significantly altered by the bivalent SA26h5-fused format (A00055; K_d ^app = 6.8 * 10“⁹ M) or the IgG 1 wild type (WT)-fused format (A00062; K_d ^app = 4.8 x 10“⁹ M). This demonstrates that these formats retain, but do not enhance, the allosteric affinity-modulating properties of the parental molecules. However, analysis of the K_d ^app for factor alpha by BLI is preferentially performed under equilibrium (steady state) binding conditions, which still accommodates concurrent changes in kinetic association (k_on) or dissociation (k_off). When analyzing these parameters for A00055 and A00062 versus A00032, the bivalent molecules revealed a considerable improvement in k_off ^app (Figure 31). This can account for the observed differences in potency in comparison to the monovalent format.

As a second example of formatting, the hPD-1 »hPD-L1 PAM CA18810 (SEQ ID NO: 11) was made in the bivalent format fused to a SA26h5 (resulting in A00265, SEQ ID NO: 73) using the framework optimized variant A00255 (SEQ ID NO: 54) and the (GGGGS)7 (SEQ ID NO: 59) linker. As shown in Figure 30E, this formatting resulted in improved potency in the SHP1 assay.

The NFAT signaling assay (as described in Example 13) more closely mimics the physiological context of a primary T cell compared to the SHP1 recruitment assay, as it involves TCR engagement. Figures 32A-B show that the bivalent SA26h5-fused hPD-1 »hPD-L1 PAM A00265 (SEQ ID NO: 73) and A00055 (SEQ ID NO: 60), as well as the trivalent A00153 (SEQ ID NO: 61), were more potent at counteracting the TCR-based activation of NFAT signaling, compared to their respective monovalent variants A00255 (SEQ ID NO: 54) and A00051 (SEQ ID NO: 74). Moreover, A00265 showed clearly improved efficacy compared to its parent, suggesting that not only the alpha factor, but also the delta factor (associated with ligand-induced receptor activation) may be influenced by the formatting, possibly through the effects of PD-1 receptor clustering, as known in the art²⁰⁵²⁰⁶, among other effects. Finally, although the IgGI WT moiety interfered partially with the assay, as shown by the decrease in signal with high doses of IgG fused irrelevant Nb (i.e. a VHH-Fc fusion of an irrelevant Nb and a hlgG1 Fc-tail) (Figure 32C), the lgG1 Fc fusion of CA19281 (SEQ ID NO: 15), A00062 (SEQ ID NO: 70), strongly improved potency in the NFAT signaling assay compared to its parent.

Therefore, formatting of PAMs of the current invention, as known in the art, is permissible and can enhance their potency in signaling assays assessing downstream effects of TCR activation, among other effects. This formatting strategy can be applied to all PAMs described herein, with representative formatted molecules and their activity listed in Table 5. The sequences of various formatted variants of the molecules of this invention encompass SEQ ID NOs 52, 60, 61 , 62, 63, 65, 67, 69, 70, 72, 73, 74, 75, or 185-239.

Table 5. Activity of various formatted modulators of this invention.

Example 23. An IgG-fused nanobody A00062 inhibits T cell activation in a mixed lymphocyte reaction (MLR).

The effect of A00062 (SEQ ID NO: 70) - a PAM of the hPD-1 »hPD-L1 complex - on T cell activation was evaluated in a mixed lymphocyte reaction (MLR) assay. In this assay, peripheral blood mononuclear cells (PBMCs) from two healthy donors were thawed and mixed at a 1 :1 cell ratio in the presence of test molecules. After five days of incubation at 37 °C with 5% CO₂, levels of IFNy and TNFa in the supernatant were measured by ELISA (LEGEND MAX™) and Luminex, respectively. In this setup, using three different donor pairs, the IgG-fused Nanobody A00062 - previously shown to stabilize the hPD-1 »hPD-L1 complex //! vitro (Example 22) - significantly reduced IFNy and TNFa release compared to both vehicle and an IgG-fused irrelevant Nanobody (Figure 33). These reductions were comparable to, or greater than, those observed with the known PD-1 agonist antibodies rosnilimab and peresolimab. Thus, T cell activation, as measured by pro-inflammatory cytokine release, was effectively suppressed by the hPD-1 »hPD-L1 complex-stabilizing Nanobody (hPD-1 »hPD-L1 PAM) of this invention.

Example 24. Nanobodies that stabilize the hPD-1 •hPD-L1 complex enable detection of the interaction between hPD-1 and hPD-L1 by microscopy.

Jurkat cells overexpressing hPD-1 were incubated in the presence or absence of 100 nM PE- conjugated hPD-L1 protein (BioTimes) in a 96-well plate. In addition, DyLight647-labeled His- tagged Nanobodies (hPD-1 »hPD-L1 complex-stabilizing CA18811 and CA19275, or an irrelevant Nanobody) or a DyLight647-labeled PD-1 antibody (peresolimab) were added to the wells. Each was pre-incubated with an anti-His antibody to confer an antibody-like structure to the Nanobodies. After a 30-minute incubation followed by a wash, images were obtained with a Leica fluorescent microscope.

As shown in Figure 34, while the anti-PD-1 antibody was detected on the surface of hPD-1- expressing cells regardless of PD-L1 presence, and the irrelevant Nanobody showed no surface binding, the hPD-1 »hPD-L1 complex-stabilizing Nanobodies CA18811 and CA19275 were only detected on the cell surface in the presence of hPD-L1 . Therefore, the hPD-1 »hPD-L1 PAMs of the current disclosure enable detection and visualization of the hPD-1 »hPD-L1 interaction by microscopy.

Example 25. Epitope characterization by BLI-based epitope binning.

To determine if the PD-1 »PD-L1 PAMs of this invention bind to overlapping or non-overlapping epitopes, epitope binning was performed employing BLI on an OCTET R8 machine. Biotinylated CA18811 , CA19275, CA19279 or CA19281 were immobilized on streptavidin (SA)-coated sensors and plunged into a solution containing the hPD-1 »hPD-L1 complex, in the presence or absence of excess PAM. Response (in nanometers, nm) was interpreted relative to a positive control lacking interference with complex formation (i.e., without VHH). In the negative control, a PAM was immobilized and incubated with the complex in the presence of the identical PAM VHH in solution, resulting in self-competition. If the VHH in solution bound to an overlapping epitope, no binding was observed, similar to the negative control. In contrast, if the signal was higher than that of the negative control - or comparable to or greater than the no-VHH condition - it indicated binding to a distinct, non-overlapping epitope. Here, each PAM was tested in both orientations, yielding two independent assessments. The conditions with immobilized CA18811 (SEQ ID NO: 12) (Figure 35A), CA19275 (SEQ ID NO: 13) (Figure 35B) and CA19281 (SEQ ID NO: 15) (Figure 35D) showed no mutual binding, resulting in signals very close to the negative control, indicating these PAMs bind to a similar (or overlapping) epitope, termed epitope A. In contrast, binding was observed for all three Nanobodies towards CA19279. Additionally, immobilized CA19279 (SEQ ID NO: 14) (Figure 35C) clearly bound to the hPD-1 »hPD-L1 complex in the presence of all three other PAMs, indicating that CA19279 binds to a distinct (non-overlapping) epitope compared to the other three PAMs, termed epitope B (Figure 35E).

Example 26. Sequence analysis.

A deep sequencing analysis was performed for optimized variants A00247-A00253, all derived from the same clonal cluster/VHH family. This enabled the identification of consensus sequences within the CDR1 , CDR2, and CDR3 regions, as shown in Figure 36A-C. Sequence logo plots were generated to visualize conserved sequence motifs across the CDRs. In these plots, the relative frequency of each amino acid at a given position is represented by the height of its one-letter code, with amino acids stacked in order of decreasing frequency. The total height of each stack reflects the information content at that position, emphasizing residues that appear more frequently than expected by chance. IMGT numbering was used for residue alignment. Sequence logos were created using PipeBio (https://pipebio.com), which enables sequence alignment and motif visualization^207,208.

Furthermore, CDR similarity was assessed and visualized through multiple sequence alignment of the non-optimized (parental) Nb sequences A00112 (SEQ ID NO: 35), A001 13 (SEQ ID NO: 36), A00114 (SEQ ID NO: 37), A001 15 (SEQ ID NO: 38), A00116 (SEQ IDNO: 39), A001 17 (SEQ ID NO: 40), A001 18 (SEQ ID NO: 41 ), A00119 (SEQ ID NO: 42), A00120 (SEQ ID NO: 43), A00121 (SEQ ID NO: 44), A00122 (SEQ ID NO: 45), A00123 (SEQ ID NO: 46), A00124 (SEQ ID NO: 47) and A00183 (SEQ ID NO: 48), which belong to the same clonal cluster. The sequence and CDR-level similarity of these molecules are illustrated in Figure 37.

Example 27. Immunization of a llama with a cross-linked hCTLA-4»hB7.1 complex to generate an antibody immune library.

The ectodomain of the human CTLA-4 (hCTLA-4 residues 37-162, SEQ ID: 243) (CTLA4, Uniprot ID: P16410-1 ) and the ectodomain of the human B7.1 (hB7.1 residues 35-242, SEQ ID: 244) (CD80, Uniprot ID: P33681 -1 ) were used to prepare a cross-linked hCTLA-4»hB7.1 complex. Both recombinant proteins were expressed in HEK293 cells in glycosylated forms. Protein purity and functionality were evaluated by SDS-PAGE and BLI on Octet (data not shown). To prepare the crosslinked hCTLA-4»hB7.1 complex for immunization, a mixture of hCTLA-4 and hB7.1 (70 pg each) was incubated with 0.05 % glutaraldehyde at 20°C in a total volume of 263 pL. The reaction was stopped after 45 minutes by the addition of 10 mM Tris-HCl pH 7.4. As illustrated in Figure 38, the level of cross-linking was monitored by SDS-PAGE and Western blot using an anti- His antibody (Biorad, MCA1396).

A llama was immunized once weekly for six consecutive weeks with a total of 50 pg of cross-linked hCTLA-4»hB7.1 complex. Following immunization, a blood sample was collected to isolate and clone a diverse repertoire of affinity-matured nanobodies specific to the hCTLA-4»hB7.1 complex. Peripheral blood lymphocytes (PBLs) were isolated from non-coagulated blood for total RNA extraction and subsequent cDNA synthesis. The resulting cDNA was used as a template to amplify the open reading frames encoding the variable domains (nanobodies) of heavy-chain antibodies. These nanobody fragments were then cloned into a suitable phage display vector for downstream screening and selection¹⁹².

Example 28. Selection of allosteric Nanobodies that bind and stabilize the hCTLA-4»hB7.1 complex.

One of the strategies we employed to identify nanobodies that bind to and stabilize the hCTLA- 4»hB7.1 complex - by targeting connective or allosteric epitopes involved in this protein-protein interaction - relied on screening immune llama libraries using phage display coupled with multiple successive rounds of biopanning, as illustrated in Figure 39.

In the first round of biopanning (Round 1 a), 1 pg of biotinylated hCTLA-4 (hCTLA-4-hFc-Avi-His; AcroBiosystems: CT4-H82F3) was immobilized on a neutravidin-coated ELISA plate in the presence of 0.4% skimmed milk in PBS to block nonspecific binding.

For this initial round of panning, the immune phage library was pre-incubated with an excess of soluble hB7.1 (0.19 pM; hB7.1 -His, AcroBiosystems: B71 -H5228) before being applied to the immobilized hCTLA-4. The mixture was incubated for 1.5 hours at 20 °C. Unbound and weakly bound phages were removed by multiple washes with PBS, and bound phages were subsequently eluted with trypsin. Eluted phages were subsequently amplified by infecting a freshly cultured E. coli strain, enabling recovery of an antibody (nanobody)-enriched repertoire specific for hCTLA-4 alone or the hCTLA-4»hB7.1 complex. Due to the presence of excess soluble hB7.1 , phages with preferential bindingto hB7.1 as a protomerwere effectively counter-selected during Round 1 a.

Phages enriched in Round 1 a served as the input for Round 1 b, where the selection process was repeated with hB7.1 and hCTLA-4 roles reversed as the immobilized and soluble proteins, respectively. 0.5 pg of biotinylated hB7.1 (hB7.1-hFc-Avi-His, AcroBiosystems: B71 -H82F2) was now immobilized on a neutravidin-coated ELISA plate in the presence of 0.4 % skimmed milk in PBS. Phages enriched in Round 1 a were then mixed with 0.7pM of soluble hCTLA-4 (hCTLA-4-His, AcroBiosystems: CT4-H5229), and next added to the immobilized hB7.1 and incubated for 1 .5 h at 20°C. Several washes with PBS were then performed prior to recovering the bound phages through trypsin digestion. Eluted phages were amplified by infecting a freshly grown E. coli culture to recover part of the Round 1 b repertoire, further enriched in Nanobodies that bind to hB7.1 alone or to the hCTLA-4»hB7.1 complex. The presence of soluble hCTLA-4 during Round 1 b effectively counter-selected phages with preferential binding to hCTLA-4 as a protomer.

The two-step procedure described in this Example comprises two rounds of biopanning designed to enrich nanobodies that specifically recognize the hCTLA-4»hB7.1 complex. Each round progressively enriches for nanobodies targeting allosteric epitopes on the complex while simultaneously counter-selecting those that bind to the soluble protomer present in excess.

Example 29. Screening for Nanobodies that stabilize the non-cross-linked hCTLA-4*hB7.1 complex by FACS.

To assess the impact of potential allosteric modulators on the dissociation constant between hCTLA-4 and its orthosteric ligand hB7.1 , a FACS-based screening assay was developed to identify nanobodies that enhance the receptor’s affinity for the ligand. To this end, Raji B cells expressing hB7.1 (Invivogen) were washed with PBS containing 2% BSA and seeded into a 96-well plate at a density of 1 x 10⁵ cells per well. The cells were incubated for 1 hour at room temperature with 2.7 nM hCTLA-4-Fc (AcroBiosystems, CT4-H5255) in the presence of tenfold diluted periplasmic extracts containing individual nanobodies. Following two washes with PBS containing 2% BSA, cells were stained with the goat anti-human IgG Fc, PE secondary antibody (Invitrogen, 12-4998-82) at a 1 :1000 dilution, followed by two additional washes. Finally, cells were resuspended in To-Pro-3 Iodide (ThermoFisher, T3605) for viability staining.

Cells were analyzed using an Attune CytPix Flow Cytometer (ThermoFisher), and data were processed with FlowJo software. For each well, a fold-change value was calculated by dividing the mean fluorescence intensity (MFI) by the average MFI of six negative control wells containing an irrelevant nanobody (Wells H3, H5, H6, H7, H8, H10). Additionally, six control wells (H1 , H2, H4, H9, H11 , H12) containing only 2.7 nM hCTLA-4-Fc, without any periplasmic extract, served as blanks. An increase in signal indicates that the nano body stabilizes the interaction between hCTLA- 4 and hB7.1 , while a decrease in signal suggests that the nanobody inhibits this interaction (Figure 40). Example 30. Validation of Nanobodies that behave as allosteric affinity modulators of the hCTLA-4»hB7.1 complex by BLI on Octet.

Example 29 illustrates the identification of several Nanobodies that stabilized the hCTLA-4»hB7.1 complex at the cell surface, as evidenced by a decreased EC₅o. In the current Example, the bimolecular equilibrium dissociation constant (K_d) of the hCTLA-4»hB7.1 interaction (in the absence of allosteric nanobodies) was compared to the affinity of hCTLA-4 for hB7.1 in the presence of saturating concentrations (2 pM) of allosteric nanobodies to estimate the cooperativity factor a, as defined herewith. For the purposes of this invention, the cooperativity factor a quantifies the binding cooperativity between an orthosteric ligand (for example hB7.1) and an allosteric modulator (nanobody) disclosed herein.

Accordingly, the biotinylated ectodomain of human hCTLA-4 (hCTLA-4-hFc-Avi-His; AcroBiosystems: CT4-H82F3, see Example 32) was immobilized on a streptavidin-coated Octet sensor. These sensors were then incubated with increasing concentrations of the hB7.1 ectodomain (hB7.1 -His; AcroBiosystems: B71 -H5228) to measure association and dissociation isotherms in the presence or absence of saturating concentrations (2 pM) of the following allosteric nanobodies: A00175 (SEQ ID NO: 245), A00176 (SEQ ID NO: 246), A00179 (SEQ ID NO: 247) and A00187 (SEQ ID NO: 248). Binding saturation curves were generated by plotting the relative increase in BLI signal amplitude against the concentration of the hB7.1 ectodomain (Figure 41). The cooperativity factor a was calculated as the ratio of the equilibrium dissociation constant (K_d) of the binary hCTLA-4»hB7.1 complex (equal to 516 nM) to the apparent K_d of the ternary complex in which the allosteric nanobody is saturated (Table 6).

Table 6. The cooperativity factor a of nanobodies A00175 (SEQ ID NO: 245), A00176 (SEQ ID NO: 246), A00179 (SEQ ID NO: 247) and A00187 (SEQ ID NO: 248). Example 31. Nanobodies as allosteric affinity modulators of the hCTLA-4»hB7.1 complex at the surface of Raji B cells expressing hB7.1.

Using the recombinant ectodomains of hCTLA-4 and hB7.1 in BLI on Octet, we validated several Nanobodies as allosteric affinity modulators of the hCTLA-4»hB7.1 complex (Example 30). To further support these findings, the ability of nanobodies to modulate the affinity of hCTLA-4 for cell- surface-expressed hB7.1 was evaluated. Raji B cells expressing hB7.1 (Invivogen) were cultured, and the binding of hCTLA-4-Fc (AcroBiosystems, CT4-H5255) was assessed by FACS in the presence or absence of allosteric nanobodies.

The different Nanobodies were purified as described previously herewith. Raji B cells expressing hB7.1 (Invivogen) were washed with PBS containing 2% BSA and seeded into a 96-well plate at 1 * 10⁵ cells per well. Cells were incubated for 1 hour at room temperature with a fixed concentration of nanobody (1 pM) and a 10-point, 3-fold serial dilution of hCTLA-4-Fc (AcroBiosystems, CT4-H5255), starting at 100 nM. Following two washes with PBS+2% BSA, cells were stained with goat anti-human IgG Fc, PE secondary antibody (Invitrogen, 12-4998-82) at a 1 :1000 dilution, followed by two additional washes. Cells were then resuspended in To-Pro3 Iodide (ThermoFisher, T3605) for viability staining. Cells were analyzed using an Attune CytPix Flow Cytometer (ThermoFisher), and data were processed with FlowJo software. Mean fluorescence intensity (MFI) values were used to determine the relative EC50 through nonlinear curve fitting using a four-parameter logistic regression model. The allosteric nanobodies reduced the EC₅₀ of hCTLA- 4 for hB7.1 compared to an irrelevant nanobody, demonstrating that these nanobodies can stabilize the hCTLA-4»hB7.1 complex at the cell surface (Table 7, Figure 42).

Table 7. Enhancement of hCTLA-4»hB7.1 complex affinity by selected Nanobodies, as determined via EC₅O analysis.

Example 32. Ligand or receptor specificity determination of hCTLA-4»hB7.1 allosteric Nanobodies. In this example, a selected allosteric nanobody A00187 (SEQ ID NO: 248) was evaluated for its binding specificity using biolayer interferometry (BLI) on an Octet-Red system (Sartorius). Binding was assessed to the hCTLA-4»hB7.1 complex, hCTLA-4 alone, hB7.1 alone, the hCTLA-4»hB7.2 complex, hB7.2 alone, the hCD28»hB7.1 complex, and the hCD28»hB7.2 complex.

Purified Nbs were biotinylated as previously described herewith. Biotinylated Nbs were loaded on streptavidin-coated biosensors (Sartorius, 18-5021 ). All the following proteins: hB7.1 (hB7.1-His, AcroBiosystems: B71 -H5228), hCTLA-4 (hCTLA-4-His, AcroBiosystems: CT4-H5229), hB7.2 (hB7.2- His, AcroBiosystems: CD6-H5223), hCD28 (hCD28, AcroBiosystems: CD8-H52H3), alone or in complex, were prepared at a concentration of 300nM. Each binding experiment was performed using the following steps: an initial 1 -minute wash in assay buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 1% BSA, 0.05% Tween-20), followed by immobilization of 1 nM biotinylated nanobody for 4 minutes. After a 1 -minute wash, the sensor was exposed to the target proteins or protein complexes for a 500-second association phase, followed by a 1 -minute dissociation step. Association (k_on) and dissociation (k_off) rates were fitted using a 1 :1 Langmuir binding model to determine the equilibrium dissociation constant K_d (Kd=k_Off/k_on).

When equimolar amounts of hCTLA-4 (the receptor) and hB7.1 (the ligand) were provided, Nanobody A00187 (SEQ ID NO: 248) was observed to bind reversibly to the complex. However, it showed no, or markedly reduced, apparent binding to the individual protomers (Figure 43).

It should be noted that the association with the hCTLA-4»hB7.1 complex is a tri-molecular process, whereas the association of the Nanobody with the separate protomers is a bi-molecular process.

Example 33. Nanobodies that behave as allosteric affinity modulators of the hCTLA-4»hB7.1 complex dampen the TCR/CD28 signaling.

The CTLA-4 Blockade Bioassay (Promega) was used to determine whether the Nb able to allosterically stabilize the CTLA-4»hB7.1 complex had a pharmacological activity. Briefly, a Jurkat cell line stably expressing human CTLA-4 and harboring a luciferase reporter under the control of a native promoter responsive to TCR/CD28 activation was co-cultured with Raji cells, which endogenously express hB7.1 and hB7.2 - ligands for CD28 and CTLA-4 - and are capable of activating the TCR. After 6 hours of co-culture in the presence of the indicated treatments, luminescence was measured according to the manufacturer’s instructions.

As expected, the CTLA-4 antagonist monoclonal antibody ipilimumab, which disrupts the interaction between CTLA-4 and its ligands B7.1 and B7.2 - thereby increasingtheir availability to engage CD28 and promote co-stimulatory signaling - resulted in a dose-dependent increase in luminescence (Figure 44). In contrast, the CTLA-4- Ig fusion protein abatacept, which blocks B7.1 and B7.2 from binding both CTLA-4 and CD28, induced a dose-dependent decrease in luminescent signal, consistent with reduced CD28-mediated co-stimulatory signaling. Under basal conditions, B7.1 and B7.2 ligands engage both CTLA-4 and CD28 receptors. Interestingly, Nanobodies A00175 (SEQ ID NO: 245) and A00179 (SEQ ID NO: 247), which stabilize the hCTLA-4»hB7.1 complex, also induced a dose-dependent decrease in the luminescent signal. This observation aligns with their ability to enhance the affinity of B7.1 for CTLA-4. In the context of this assay - where B7.1 can interact with both its receptors - these Nanobodies preferentially shift ligand binding toward the inhibitory CTLA-4 pathway, thereby reducing CD28-mediated co-stimulation.

Example 34. A Nanobody that behaves as an allosteric affinity modulator of the hCTLA-4*hB7.1 complex reduces B7.1-induced T cell activation.

Frozen CD4⁺ T cells, isolated from a healthy donor, were thawed and seeded into a 96-well plate for overnight recovery. The following day, T cells were stimulated using Epoxy M-280 Dynabeads (Invitrogen) at a 4:1 bead-to-cell ratio. Beads were coated either with lysozyme alone (negative control) or with an anti-CD3 antibody (Invitrogen) to triggerTCR activation. In addition to stimulation with anti-CD3-coated beads, T cells were further activated using 3 pg/mL of B7.1-Fc (AcroBiosystems: B71 -H5259) or 250 ng/mL of anti-CD28 antibody (Invitrogen) as a control. Test molecules were co-incubated in the condition containing both anti-CD3 beads and B7.1 -Fc. After 3 days of incubation, the expression of CD25 was analyzed by FACS, using a Brilliant Violet 421 - tagged antibody (BioLegend).

As shown in Figure 45, stimulation with anti-CD3 beads induced an increase in CD25 expression compared to control beads, which was further enhanced by co-treatment with either anti-CD28 or B7.1-Fc. An irrelevant Nanobody had no effect on the baseline activation observed with anti-CD3 plus B7.1 -Fc. In contrast, both abatacept (at 100 nM) and the CTLA-4»hB7.1 complex-stabilizing Nanobody A00175 (at 600 and 120 nM) reduced CD25 expression to levels comparable to anti-CD3 stimulation alone, demonstrating their capacity to counteract the co-stimulatory effect of B7.1 -Fc.

While abatacept likely causes the observed decrease by directly blocking B7.1 binding to CD28, the effect of Nanobody A00175 may be attributed to its enhancement of B7.1 affinity for CTLA-4. This increased affinity could either directly activate the inhibitory CTLA-4 signaling pathway or competitively prevent the stimulatory CD28 receptor from engaging its ligands. In either scenario, these findings demonstrate that a Nanobody stabilizing the hCTLA-4»hB7.1 complex can effectively reduce human T cell activation, as indicated by decreased CD25 expression.

Example 35. Immunization of a llama with a cross-linked hCD200R»hCD200 complex to generate an antibody immune library. The ectodomain of human CD200R1 (hCD200R1 , also referred to as hCD200R throughout this disclosure, UniProt ID: Q8TD46), composed of residues 27-266 (SEQ ID NO: 249) and the ectodomain of human CD200 (hCD200, UniProt ID: P41217), composed of residues 31-232 (SEQ ID NO: 250), were used to prepare a cross-linked hCD200R»hCD200 complex. Both recombinant proteins were expressed in HEK293 cells and are glycosylated. Protein purity and functionality were assessed by SDS-PAGE and Bio-Layer Interferometry (BLI) on the Octet instrument (data not shown).

To identify suitable conditions to covalently stabilize the hCD200R»hCD200 complex, glutaraldehyde was used as a chemical cross-linker in the concentration range from 0.3% to 0% (Figure 46). hCD200R, hCD200 or the hCD200R»hCD200 complex was incubated with the respective concentrations of glutaraldehyde at 4°C in a total volume of 20 pl. The reaction was stopped after 30 minutes by the addition of 50 mM Tris-HCl, pH 7.4. The level of cross-linking was monitored by SDS-PAGE on precast gradient SDS-PAGE gels (Biorad, mini-protean TGX) stained with Instant-Blue Coomassie protein stain (Abeam, ab119211 ), and anti-His western blot using 0.2 pm transblot mini PVDF membrane (Biorad, 1704156) and a mouse anti-Histidine tag antibody (Biorad, MCA1396). As shown, the addition of glutaraldehyde causes the formation of a higher molecular weight band correspondingto the cross-linked hCD200R»hCD200 complex.

To prepare the cross-linked hCD200R»hCD200 complex for immunization, a mixture of 250 pg of recombinant hCD200R (Aero Biosystems, CR2-H52H6) and 250 pg of recombinant hCD200 (Aero Biosystems, OX2-H5228) was incubated with 0.02% glutaraldehyde at 4 °C in a total volume of 1054 pL. The reaction was stopped after 30 minutes by addition of 20 mM Tris-HCl pH 7.4.

Two llamas were immunized every two weeks for a total of four immunizations, using 100 pg of the cross-linked hCD200R»hCD200 complex per immunization. Following the final boost, peripheral blood was collected to isolate lymphocytes for the generation of a Nanobody library. This library was constructed to enable the cloning and selection of a diverse repertoire of affinity-matured Nanobodies specifically recognizing the hCD200R»hCD200 complex. Next, peripheral blood lymphocytes (PBLs) were isolated from the noncoagulated blood of the llamas for the purification of total RNAand the synthesis of cDNA. This cDNA served as a template to amplify the open reading frames codingforthe variable domains (Nanobodies) of the heavy-chain antibodies and Nanobody fragments were then cloned into appropriate phage display vectors, as described herewith.

For this project, from each llama immunized, two libraries containing different tags were constructed, namely: Triple-myc-his tag (SEQ ID NO: 251 ,

AAAEQKLISEEDLEQKLISEEDLEQKLISEEDLGAAHHHHHH) or his-EPEA-tag (SEQ ID NO: 252, HHHHHHEPEA). Selections were performed in parallel on both types of libraries (as described herewith) and output sequencing identified sequences from both libraries belonging to the same sequence cluster. This convergence strongly suggests that the selection of PAMs was driven by their specific binding properties to the hCD200R»hCD200 complex rather than by differences in tag sequences present in the respective libraries.

Example 36. Selection of allosteric Nanobodies that bind and stabilize the hCD200R*hCD200 complex.

Phage display was performed using the constructed llama immune libraries, followed by successive rounds of biopanning, as illustrated in Figure 47, to discover VHHs that bind to and stabilize the hCD200R»hCD200 complex by binding to connective or allosteric epitopes of this protein- protein interaction.

In a first round (Round 1 a) we immobilized 0.2 pg of biotinylated hCD200R (hCD200R-Fc-Avi, AcroBiosystems: CR2-H82F4) or 0.1 pg of biotinylated hCD200 (hCD200-Fc-Avi, AcroBiosystems: OX2-H82F1) on a neutravidin-coated ELISA plate in the presence of 0.4 % of skimmed milk in PBS.

For the first round of panning, the immune library was mixed with an excess of 2 pM soluble hCD200 (hCD200-His, AcroBiosystems: OX2-H5228) or 1.5 pM hCD200R (hCD200R-His, AcroBiosystems: CR2-H52H6), and then added to immobilized hCD200R or hCD200, respectively, and incubated for 1 .5 hours at 20 °C. Then, several washes with PBS were performed before recovering the bound phage by trypsin digestion. Eluted phages were then amplified by infecting freshly grown E. coli to recover part of the initial repertoire enriched in VHHs that bind to hCD200R alone, hCD200 alone, or the hCD200R»hCD200 complex. Due to the excess of soluble complex partner in solution, phages preferentially binding to the individual protomers were effectively counter-selected in Round 1 a.

Phages enriched in Round 1 a were subsequently used as input for selection Round 1 b, following the same procedure but with the immobilized and soluble proteins swapped between hCD200R and hCD200 for each condition. Specifically, 0.1 pg of biotinylated hCD200 (hCD200-Fc-Avi, AcroBiosystems: OX2-H82F1) or 0.2 pg of biotinylated hCD200R (hCD200R-Fc-Avi, AcroBiosystems: CR2-H82F4) was immobilized on neutravidin-coated ELISA plates in PBS containing 0.4% skimmed milk. The phages were incubated with an excess (0.5 pM) of soluble hCD200R (hCD200R-His, AcroBiosystems: CR2-H52H6) or soluble hCD200 (hCD200-His, AcroBiosystems: OX2-H5228), depending on the selection condition. This mixture was then added to wells coated with the immobilized respective complex partner and incubated for 1.5 hours at 20 °C. Following multiple PBS washes, bound phages were eluted by trypsin digestion. The eluted phages were amplified by infecting a freshly grown E. coli culture, thus recovering the Round 1 b repertoire further enriched in VHHs specific to hCD200R, hCD200, or the hCD200R»hCD200 complex. As in Round 1a, phages with preference for individual protomers were counter-selected through the presence of an excess of complex partner in solution.

The two-step procedure utilized in this Example involves two successive rounds of biopanning designed to enrich for VHHs that specifically recognize the hCD200R»hCD200 complex. Each round of the biopanning contributes to cumulatively select for VHHs targeting allosteric epitopes within the complex, while simultaneously counter-selecting those that preferentially bind to the individual protomers present in excess in solution.

Example 37. Screening for Nanobodies that stabilize the non-cross-linked hCD200R*hCD200 complex by ELISA.

To evaluate the effect of potential allosteric modulators on the dissociation constant between the receptor hCD200R and its orthosteric ligand hCD200, we developed an ELISA protocol analogous to that described in Example 6, aimed at identifying Nanobodies that enhance complex stability. To screen for such potential stabilizers of the hCD200R»hCD200 complex, ten individual clones were selected based on sequence analysis (data not shown), expressed in TG-1 cells, and purified by Immobilized Metal Affinity Chromatography (IMAC). For ELISA, 0.08 pg of hCD200-Fc (human CD200-Fc-tag, AcroBiosystems, OX2-H5251 ) was coated on ELISA plates. The coated wells were then incubated with sub-saturating amounts (0.05 pg) of biotinylated hCD200R-Fc-Avi (human CD200R, Fc-tag, Avi-tag, AcroBiosystems, CR2-H82F4) together with 1 pM of each purified individual Nanobody. After washing, the plates were developed using a streptavidin-alkaline phosphatase conjugate (Streptavidin Alkaline Phosphatase, Promega, V5591 ). Irrelevant VHH samples, which do not interfere with complex formation, were used as reference controls. A fold change value for each well was calculated using the observed optical density (CD) divided by the average CD of the reference containing an irrelevant VHH.

An increase in signal indicates that the Nanobody stabilizes the interaction between hCD200R and hCD200, while a decrease in signal suggests that the Nanobody inhibits this interaction (Figure 48). Nanobodies A00267 (SEQ ID NO: 253), A00268 (SEQ ID NO: 254), A00269 (SEQ ID NO: 255), A00270 (SEQ ID NO: 256), A00271 (SEQ ID NO: 257), A00274 (SEQ ID NO: 258), A00276 (SEQ ID NO: 259), and A00277 (SEQ ID NO: 260) are thus identified as candidate positive allosteric modulators (PAMs) of the hCD200R»hCD200 complex.

Example 38. Characterization of purified Nanobodies binding to hCD200R, hCD200 or the cross-linked hCD200R»hCD200 complex by ELISA. Purified Nbs were tested for binding to hCD200R alone, hCD200 alone or to the cross-linked complex of hCD200R»hCD200 using a typical ELISA protocol. To this end, 0.1 pg of hCD200R-His (AcroBiosystems, CR2-H52H6), hCD200-His (AcroBiosystems, OX2-H5228) or the cross-linked complex of hCD200R»hCD200 were immobilized on Nunc™ Immuno Maxisorp ELISA plates (Thermo Scientific, 439454), while non-coated wells served as a control. Following a blocking step with 4% milk in PBS, wells were incubated with 2 pM of purified Nb for l hour. The ELISA plates were developed using a mouse anti-cMyc primary antibody (Merck, 11667149001), followed by a goat anti-mouse IgG alkaline phosphatase-conjugated secondary antibody (Sigma-Aldrich, A3562). For each coated antigen, an irrelevant Nanobody was used as a reference negative control. Accordingly, a fold-change in signal was calculated for each tested VHH by dividing its optical density (OD) by the OD of the corresponding irrelevant Nanobody control (Figure 49). Clones showing a fold change of two or greater were considered to bind the respective coated antigen. Based on this analysis, three distinct binding profiles were identified. While A00267 (SEQ ID NO: 253), A00268 (SEQ ID NO: 254), A00269 (SEQ ID NO: 255), A00270 (SEQ ID NO: 256), and A00271 (SEQ ID NO: 257) bind both hCD200R-His and the hCD200R»hCD200 complex, A00272 (SEQ ID NO: 261 ) and A00273 (SEQ ID NO: 262) bind to hCD200-His and the complex. In contrast, A00274 (SEQ ID NO: 258), A00276 (SEQ ID NO: 259), and A00277 (SEQ ID NO: 260) bind exclusively to the hCD200R»hCD200 complex and not to either of the individual protomers.

Example 39. Nanobodies that act as allosteric affinity modulators of the hCD200R»hCD200 complex.

From Examples 37 and 38, we conclude that several Nanobodies predominantly bind to the hCD200R»hCD200 complex rather than to the individual protomers, hCD200R or hCD200 - similar to the observations made in Example 7 for the hPD-1 »hPD-L1 complex. However, comparing the binding of an antibodyto a binary complex (hCD200R»hCD200) versus its binding to the individual protomers (hCD200R or hCD200 alone) is challenging, as it requires contrastingthe properties of ternary complexes with those of binary complexes. To exemplify and validate the stabilization effect, we selected two PAMs representing distinct binding profiles (see the previous Example): A00267, which binds both CD200R and the hCD200R»hCD200 complex, and A00276, which binds only to the hCD200R»hCD200 complex but not to the individual protomers. Factors a of these two PAMs were determined. Using the methods described herewith, a titration series of hCD200 for hCD200R binding was performed by BLI in the presence of saturating concentrations of the allosteric Nanobodies. The factor a determined from this experiment quantifies the binding cooperativity between the orthosteric ligand (hCD200) and the positive allosteric modulating Nanobody towards the receptor CD200R. Accordingly, we immobilized the biotinylated ectodomain of hCD200R-Fc (Human CD200R, Fc-tag, Avi-tag, AcroBiosystems, CR2-H82F4) on a neutravidine-coated ELISA plate. The sensors were plunged into a titration series of hCD200-His (human CD200-His, AcroBiosystems, OX2-H5228) in the presence of 2 pM of a test Nb or an irrelevant Nb. K_d ^app and factors awere calculated as described in Example 8 and are summarized in Figure 50.

Example 40. Validation of molecules with a positive allosteric modulator profile towards the hCD200R«hCD200 complex by ELISA.

To further validate the allosteric complex stabilization effect observed with hCD200R»hCD200 PAMs of this disclosure, we compared the EC₅₀ values obtained from an hCD200R titration series for hCD200 binding in the presence of saturating concentrations of selected allosteric Nanobodies to the EC₅₀ value measured in the presence of an irrelevant Nanobody, using ELISA. This comparison was used to determine the cooperativity factor a. Herein, factor a is the measure of binding cooperativity between the orthosteric ligand (hCD200) and the positive allosteric modulating Nanobody towards the receptor CD200R.

Accordingly, we immobilized 0.08 pg of the ectodomain of hCD200-Fc (Human CD200, Fc-tag, AcroBiosystems, OX2-H5251) on a neutravidine-coated ELISA plate. After blockingwith 4 % milk in PBS, the wells were incubated with a titration series of biotinylated hCD200R-Fc-Avi (human CD200R-Fc-Avi, AcroBiosystems, CR2-H82F4) in the presence of 2 pM of allosterically modulating or irrelevant VHH. After washing, plates were developed with a streptavidin-alkaline phosphatase conjugate (Streptavidin Alkaline Phosphatase, Promega, V5591).

The generated dose-response curves illustrate the concentration-dependent changes in complex formation (Figure 51). The dose-response curves were subjected to non-linear regression fitting to estimate EC₅o values. EC₅₀ values calculated from curves with the allosteric or irrelevant VHH enabled estimation of the cooperativity factor a, as shown in Figure 51.

Example 41. Immunization of a llama with a cross-linked hTIGIT»hPVR complex to generate an antibody immune library.

The ectodomain of the human T cell immunoreceptor with Ig and ITIM domains (hTIGIT, Uniprot ID: Q495A1 , residues 22-141 , SEQ ID: 265) and the ectodomain of the human poliovirus receptor (hPVR, Uniprot ID: PI 5151 , residues 21 -343, SEQ ID:266) were used to prepare a cross-linked hTIGIT»hPVR complex. Both recombinant proteins were expressed in HEK293 cells and are glycosylated. Protein purity and functionality were evaluated by SDS-PAGE and BLI on Octet (data not shown). To prepare crosslinked the hTIGIT*hPVR complex for immunization, a mixture of 250 pg of each hTIGIT (Aero Biosystems, TIT-H52H5) and hPVR (Aero Biosystems, CD5-H5223) was incubated with 0.1 % glutaraldehyde at 4°C in a total volume of 701 pL. The reaction was stopped after 30 minutes by the addition of 20 mM Tris-HCl pH 7.4. As illustrated in Figure 52, the level of cross-linking was monitored by SDS-PAGE on precast gradient SDS-PAGE gels (Biorad, Mini-protean TGX) stained with the Instant-Blue Coomassie protein stain (Abeam, ab119211), and anti-His western blot using an anti-His antibody (Biorad, MCA1396).

Two llamas were immunized at two-week intervals for a total of four times, using 100 pg of crosslinked hTIGIT»hPVR complex. After immunization, a blood sample was collected to clone a diverse set of affinity matured Nanobodies specific to the hTIGIT*hPVR complex. Peripheral blood lymphocytes (PBLs) were isolated from the noncoagulated blood for the purification of total RNA and the synthesis of cDNA. This cDNA served as a template to amplify the open reading frames coding for the variable domains (Nanobodies) of heavy-chain antibodies and Nanobody fragments were then cloned into an appropriate phage display vector¹⁹².

Example 42. Selection of allosteric Nanobodies that bind and stabilize the hTIGIT»hPVR complex.

We combined phage display from the constructed Llama immune libraries with successive biopanning steps, as illustrated in Figure 53, to discover VHHs that bind to and stabilize the hTIGIT»hPVR complex by binding to connective or allosteric epitopes of this protein-protein interaction.

In a first round (Round 1 a) we immobilized 0.25 pg of biotinylated hTIGIT (hTIGIT-hFc-Avi, AcroBiosystems: TIT-H82F-1 ) or biotinylated hPVR (CD155 (PVR)-hFc-Avi, AcroBiosystems: CD5- H82F6) on a neutravidin-coated ELISA plate in the presence of 0.4 % of skimmed milk in PBS.

For the first round of panning, the immune library was mixed with an excess (0.5 pg) of soluble hPVR (CD155 (PVR)-hFc, AcroBiosystems: CD5-H5251) or hTIGIT (hTIGIT-hFc, AcroBiosystems: TIT- H5254), and then added to the immobilized hTIGIT or hPVR, respectively, and incubated for 1.5 hours at 20 °C. Following, several washes with PBS were performed before recovering the bound phage by trypsin digestion. Eluted phages were then amplified by infecting freshly grown E. coli to recover part of the initial repertoire enriched in VHHs that bind to hTIGIT alone, hPVR alone or to the hTIGIT* hPVR complex. Because of the excess of soluble protein complex partner in solution, phage preferentially binding to the individual protomers were counter-selected in Round 1 a.

Phages enriched in Round 1 a were subsequently used as the input of selection Round 1 b, repeating the process but inversing hTIGIT and hPVR as the immobilized and the soluble protein for the respective selection condition. 0.25 pg of biotinylated hPVR (CD155 (PVR)-hFc-Avi, AcroBiosystems: CD5-H82F6) or biotinylated hTIGIT (hTIGIT-hFc-Avi, AcroBiosystems: TIT-H82F-1) was now immobilized on a neutravidin-coated ELISA plate in presence of 0.4% skimmed milk in PBS.

The phages were incubated with an excess (0.5 pg) of soluble hTIGIT (hTIGIT-hFc, AcroBiosystems: TIT-H5254) or soluble hPVR (CD155 (PVR)-hFc, AcroBiosystems: CD5-H5251), respectively, and next added to the immobilized complex partner and incubated for 1 .5 hours at 20 °C. Then several washing steps were performed with PBS before recovering the bound phage after trypsin digestion. Eluted phages were next amplified by infecting a freshly grown E. coli culture to recover part of the Round 1 b repertoire, further enriched in VHHs that bind to hTIGIT alone, hPVR alone or to the hTIGIT»hPVR complex. As in Round 1 a, phages with preference for individual protomers were counter-selected through the presence of an excess complex partner in solution.

The two-step procedure described in this example consists of two successive rounds of biopanning aimed at enriching for VHHs that specifically recognize the hTIGIT»hPVR complex. Each round of the biopanning contributes to cumulatively select for VHHs targeting allosteric epitopes within the complex, while simultaneously counter-selecting those that preferentially bind to the individual protomers present in excess in solution.

Example 43. Screening for Nanobodies that stabilize the non-cross-linked hTIGIT*hPVR complex by ELISA.

To evaluate the effect of potential allosteric modulators on the dissociation constant between the receptor hTIGIT and its orthosteric ligand hPVR, we developed an ELISA protocol analogous to that described in Example s, aimed at identifying Na nobodies that enhance complex stability. To screen for potential stabilizers of the hTIGIT»hPVR complex, individual clones were cultured in a 96-well format, induced with isopropyl 0-D-1 -thiogalactopyranoside (IPTG), and periplasmic extracts were prepared following previously described protocols. To perform the ELISA, we coated 0.25 pg of hPVR-hFc (Human CD155/ PVR Protein, Fc-tag, AcroBiosystems, CD5-H5251 ) on ELISA plates and incubated the coated wells with sub-saturating amounts (1.5 ng) of biotinylated hTIGIT-Avi-His (AcroBiosystems, TIT-H82E6) together with periplasmic extracts containing individual Nanobodies. After washing, plates were developed with a streptavidin-alkaline phosphatase conjugate (Streptavidin Alkaline Phosphatase, Promega, V5591).

Row H of the ELISA plate contained periplasmic extracts of irrelevant VHHs, which do not interfere with complex formation. These served as reference values to identify hits, defined as signals exceeding three times the standard deviation of the reference. A fold change value for each well was calculated using the observed optical density (OD) divided by the average OD of the reference containing an irrelevant Nb. An increase in signal indicates that the Nanobody stabilizes the interaction between hTIGIT and hPVR (Figure 54).

Example 44. Characterization of purified VHHs binding to hTIGIT, hPVR or the cross-linked hTIGIT-hPVR complex by ELISA.

To characterize potential binders to hTIGIT, hPVR, or their complex, and to prioritize candidate PAMs, selected clones were expressed in TG-1 cells and purified using IMAC. The purified VHHs were tested for binding to hTIGIT alone, hPVR alone or to the cross-linked complex of hTIGIT*hPVR using a standard ELISA protocol. To this end, 0.1 pg of hTIGIT-hFc (AcroBiosystems, TIT-H5254), hPVR-hFc (Human PVR protein, Fc-tag, AcroBiosystems, CD5-H5251) or the cross-linked complex of hTIGIT»hPVR were immobilized on Nunc™ Immuno Maxisorp ELISA plates (Thermo Scientific, 439454), while non-coated wells served as a control. Following blocking with 4% milk in PBS, wells were incubated for 1 hour with 1 pM of purified VHH. The ELISA plates were developed using a mouse anti-cMyc primary antibody (Merck, 11667149001), followed by a goat anti-mouse IgG alkaline phosphatase-conjugated secondary antibody (Sigma Aldrich, A3562). For each coated antigen, an irrelevant VHH was used as reference negative control. Consequently, for each VHH, a fold change of signal is calculated by dividing the optical density (OD) in the presence of the VHH by the OD of the corresponding reference negative control. (Figure 55).

Clones displaying a fold change of two or greater were classified as binders to the respective coated antigen. As a representative example, A00314 (SEQ ID NO: 264) binds both hTIGIT-hFc and the hTIGIT»hPVR complex, whereas A00312 (SEQ ID NO: 263) binds exclusively to the hTIGIT*hPVR complex and not to the individual protomers.

Example 45. Validation of selected VHHs exhibiting a positive allosteric modulator profile for the hTIGIT»hPVR complex using ELISA.

From Examples 43 and 44, we concluded that Nanobodies A00314 (SEQ ID NO: 264) and A00312 (SEQ ID NO: 263) act as stabilizers of the hTIGIT* hPVR complex and exhibit distinct binding profiles. Specifically, A00314 binds both hTIGIT and the hTIGIT*hPVR complex, whereas A00312 binds exclusively to the hTIGIT*hPVR complex. To validate their stabilizing effects, we performed a titration series of hTIGIT for hPVR binding in the presence of saturating concentrations (1 pM) of A00314 or A00312, and compared the resulting EC₅o values to those obtained in the presence of an irrelevant VHH using ELISA. The ratio of these EC₅o values was used to estimate the cooperativity factor (a), which serves as a quantitative measure of binding cooperativity between the orthosteric ligand (hPVR) and the positive allosteric modulator (nanobody). Accordingly, 1 g of the ectodomain of hPVR-hFc (Human CD155 / PVR Protein, Fc-tag, AcroBiosystems, CD5-H5251) was immobilized on a neutravidin-coated ELISA plate. After blocking with 4% milk in PBS, wells were incubated with a titration series of biotinylated hTIGIT-Avi-His (AcroBiosystems, TIT-H82E6) in the presence of 1 pM of either allosteric modulating or irrelevant VHH. Following washing steps, plates were developed using a streptavidin-alkaline phosphatase conjugate (Promega, Streptavidin Alkaline Phosphatase, V5591).

The generated dose-response curves illustrate the concentration-dependent changes in complex formation (Figure 56). The dose-response curves were subjected to non-linear regression fitting to estimate EC50 values. EC₅₀ values calculated from curves with allosteric or irrelevant VHH enabled estimation of the cooperativity factors a, as shown in (Figure 56).

Example 46. Epitope mapping of hPD-1 »hPD-L1 complex PAM Nbs through screening of a PD- 1 mutant library.

To pinpoint amino acid residues that are part of the epitope recognized by our panel of hPD-1 »hPD- L1 stabilizing nanobodies (Nbs), we employed an ELISA-based mutagenesis approach. The coding sequence of the extra-cellular domain of hPD-1 of SEQ ID: 08 (AA33 to AA150) was cloned into a Golden Gate variant of the pMESy4 vector (Genbank KF415192.1 ), enabling expression of recombinant EPEA-tagged hPD-1 in the periplasmic space of the E.coli cells. To generate a diverse pool of hPD-1 mutants, we performed error-prone PCR on the hPD-1 gene as previously described²⁰⁹, introducing random point mutations throughout the coding region. Further mutations were created by site-directed mutagenesis. All mutant genes were ligated into the vector using the Golden Gate cloning strategy²¹⁰ and transformed into TG1 E. coli cells. To express hPD-1 mutant proteins in small-scale E. coli cultures, TG1 cells harboring the different plasmids were grown separately and induced with IPTG, followed by an overnight incubation. Cell pellets were harvested and stored at -20 °C to facilitate protein extraction upon thawing. This generated a library of hPD-1 mutants that were screened for their ability to interact with hPD-1 »hPD-L1 positive allosteric modulator nanobodies CA19275 (SEQ ID NO: 13), CA19279 (SEQ ID NO: 14) and CA18811 (SEQ ID NO: 12) in complex with hPD-L1 .

Nanobodies were engineered to include an AviTag and a His-tag sequence (SEQ ID NO: 240) at the C-terminus to facilitate site-specific biotinylation. The expression vector was designed to coexpress the birA gene, enabling in vivo biotinylation of the AviTag recombinant protein expression. Biotinylated nanobodies were purified and immobilized on neutravidin-coated Nunc-immuno maxisorp ELISA plates (Thermo Scientific, 439454) at a concentration of 6 pg/ml. Following their immobilization, nanobodies CA19275, CA19279, or CA18811 were incubated with the same batch of bacterial extracts containing the hPD-1 mutant proteins in the presence of 0.5 pM of hPD-L1 (Aero Biosystems, PD1-H5229). After incubation, the binding of the hPD-1 WT or mutant proteins on the immobilized Nb was quantified via detection of the C-terminal EPEA tag of recombinantly expressed hPD-1 clones, as in a sandwich ELISA.

Interestingly, the signal of the wild-type (WT) hPD-1 binding to CA19279 was always about 2-fold higher than of CA18811 or CA19275. Therefore, the analysis was performed using the fold change between mutant and WT signals per each Nb. After this normalization, a clone for a hPD-1 mutant protein which showed no binding difference between each Nb, is identified as being neutral, i.e. not part of either epitope A or B. Conversely, considering that CA18811 and CA19275 bind “Epitope A” while CA19279 binds a non-overlapping “Epitope B” (see Example 25), mutations that disrupt the “Epitope B” should reduce the mutant’s retention on CA19279, but not on CA19275 and CA18811 . This was the case for the mutations E141 K and V43I (Figure 57). Both the E141 and V43 residues are situated within the “Epitope B” identified by analysis of the structure obtained in Example 10 (Figure 13), which confirmed the validity of the method. Through the same analysis, the mutations E61 K and S62K were found to disrupt binding for CA19275 and CA18811 , but not CA19279. As can be seen in Figure 57, E61 and S62 are located near the interface between hPD-1 and hPD-L1 , which is in line with the Epitope A being a “connective epitope”, i.e. spanning the interface between hPD-

1 and hPD-L1.

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Claims

1 . An allosteric modulator of a protein complex, wherein the protein complex comprises at least one immunoinhibitory surface receptor and at least one ligand, and wherein the allosteric modulator comprises an immunoglobulin single variable domain (ISVD) which specifically binds to an epitope predominantly present on said protein complex, and wherein said modulator, upon specific binding, is an inducer of positive cooperativity.

2. The allosteric modulator of claim 1 , wherein the protein complex comprises at least one immune receptor set forth in Figure 1 and at least one ligand of said immune receptor set forth in Figure 2.

3. The allosteric modulator of claim 2, wherein the immune receptor of Figure 1 and the ligand of Figure 2 are provided as protein- protein interactors in Figure 3.

4. The allosteric modulator of claims 1 -3, wherein positive cooperativity is defined by a factor a value higher than 1 .

5. The allosteric modulator of claim 4, wherein positive cooperativity is additionally defined by a Hill coefficient value higher than 1 .

6. The allosteric modulator according to any one of claims 1-5, wherein the modulator, upon binding to said protein complex, modulates downstream signaling mediated by one or more proteins of the complex.

7. The allosteric modulator according to any one of claims 1 -6, wherein the modulator has spatiotemporal selectivity for the protein complex.

8. The allosteric modulator according to any one of claims 1 -7, wherein said allosteric modulator binds to an epitope that is at least partially located on one immune receptor and/or at least partially located on one ligand engaged in said protein complex.

9. The allosteric modulator according to claims 1 -8, wherein said allosteric modulator is an antibody, an active antibody fragment, an immunoglobulin single variable domain (ISVD), a single domain antibody, a VHH, or a nanobody (Nb).

10. The allosteric modulator according to any one of claims 1 -9, further comprising a functional moiety, preferably selected from a therapeutic moiety and/or a half-life extending moiety.

11 . The allosteric modulator of a protein complex according to any one of claims 1 -10, which is a multivalent and/or multispecific modulator, preferably comprising at least two moieties capable of bindingto an epitope predominantly present on said protein complex, and wherein said epitope is at least partially located on one immune receptor and/or at least partially located on one ligand of said protein complex.

12. A nucleic acid molecule encoding the allosteric modulator of a protein complex according to any of the preceding claims.

13. Avector comprising the nucleic acid molecule according to claim 12.

14. A pharmaceutical composition comprising the allosteric modulator of any one of claims 1 to 11 , the nucleic acid molecule of claim 12, or the vector of claim 13.

15. The allosteric modulator of any one of claims 1 to 11 , the nucleic acid molecule of claim 12, the vector of claim 13, or the pharmaceutical composition of claim 14, for use as a medicament.

16. The allosteric modulator of any one of claims 1 to 11 , the nucleic acid molecule of claim 12, the vector of claim 13, or the pharmaceutical composition of claim 14, for use in treatment of an autoimmune disease.

17. The allosteric modulator of any one of claims 1 to 11 , the nucleic acid molecule of claim 12, the vector of claim 13, or the pharmaceutical composition of claim 14, for use in treatment of graft-versus-host disease (GVHD).

18. The allosteric modulator of any one of claims 1 to 11 , the nucleic acid molecule of claim 12, the vector of claim 13, or the pharmaceutical composition of claim 14, for use in treatment of an allergic disease.

19. The allosteric modulator according to anyone of claims 1 to 11 , wherein the modulator is fused or conjugated to a detection moiety, preferably wherein said detection moiety is a label ora tag.

20. A method for detecting a protein complex comprising at least one immunoinhibitory surface receptor and at least one ligand, wherein the method comprises contacting a sample with the allosteric modulator of claim 19.

21 . The allosteric modulator of any one of claims 1 -11 or of claim 19, the nucleic acid molecule of claim 12, the vector of claim 13, or the pharmaceutical composition of claim 14, for use as a diagnostic.

22. A method for producing an allosteric modulator of a protein complex according to any one of claims 1 -11 , the method comprising the steps of: a. selecting, from a collection of candidate modulators, a modulator or a plurality of modulators that specifically bind to an epitope predominantly present in said protein complex, and b. identifying, among the selected modulator(s) in a), the allosteric modulator(s) of said protein complexthat induce positive cooperativity to the protein complex upon bindingto it, c. and optionally, identifying, among the selected modulator(s), allosteric modulator(s) of said protein complex that are capable of modulating downstream signaling mediated by one or more proteins of the complex.

23. The method according to claim 22, wherein the positive cooperativity is determined by a factor avalue higherthan 1 .

24. The method according to claim 23, wherein the positive cooperativity is additionally determined by a Hill coefficient value higherthan 1.

25. The method according to claims 22-24, wherein said allosteric modulator specifically binds to an epitope predominantly present in said protein complex, and wherein said epitope is at least partially located on one immune receptor and/or at least partially located on one ligand of said protein complex.

26. The method according to any one of claims 22-25, wherein said collection of candidate modulators is a library comprising: antibodies, active antibody fragments, immunoglobulin single variable domains (ISVDs), single domain antibodies, VHHs, or nanobodies (Nbs).

27. The method of claim 26, wherein said antibodies, active antibody fragments, ISVDs, single domain antibodies, VHHs, or Nbs are obtained from immunization of a non-human animal with said protein complex in a cross-linked form, preferably wherein the non-human animal belongs to the biological family Camelidae.

28. The method of claims 22-27, wherein said selection of modulator(s) from a collection of candidate modulators in step a) involves displaying candidate modulators on a cell surface via display biopanning, including phage display, yeast display, or mammalian display.