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WO2026008665A1 - Liants du complexe pd-1•pd-l1 et leur utilisation - Google Patents

Liants du complexe pd-1•pd-l1 et leur utilisation

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Publication number
WO2026008665A1
WO2026008665A1 PCT/EP2025/068744 EP2025068744W WO2026008665A1 WO 2026008665 A1 WO2026008665 A1 WO 2026008665A1 EP 2025068744 W EP2025068744 W EP 2025068744W WO 2026008665 A1 WO2026008665 A1 WO 2026008665A1
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WIPO (PCT)
Prior art keywords
comprises seq
hpd
complex
binding
seq
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Inventor
Jan Steyaert
Alexandre WOHLKÖNIG
Thomas ZÖGG
Marie MIGLIANICO
Toon Laeremans
Sarah TRIEST
Isabelle CAMBRÉ
Catelijne Stortelers
Els Pardon
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Vlaams Instituut voor Biotechnologie VIB
Vrije Universiteit Brussel VUB
Original Assignee
Vlaams Instituut voor Biotechnologie VIB
Vrije Universiteit Brussel VUB
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Publication of WO2026008665A1 publication Critical patent/WO2026008665A1/fr
Pending legal-status Critical Current
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/32—Immunoglobulins specific features characterized by aspects of specificity or valency specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • This disclosure relates to an immunoglobulin single variable domain (ISVD)-containing modulator that allosterically binds to a three-dimensional (3D) epitope present on the human PD-1 »PDL1 (hPD-1 »hPD-L1 ) complex.
  • the modulator regulates cooperativity within this complex, thereby affecting the binding affinity between hPD-1 and hPD-L1 and/or downstream signaling pathways.
  • the allosteric modulator of this invention selectively induces positive cooperativity in the hPD-1 »hPD-L1 complex, thus suppressing immune responses in a spatiotemporally restricted manner. Accordingly, such allosteric modulators are useful as therapeutic agents for inflammatory diseases, such as autoimmune diseases, allergic diseases, or graft-versus-host disease (GVHD).
  • inflammatory diseases such as autoimmune diseases, allergic diseases, or graft-versus-host disease (GVHD).
  • 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 .
  • 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 molecules 6 . 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 effects 7 .
  • An inhibitory immune receptor transmits intracellular signaling upon binding with its orthosteric ligand(s), leading to the formation of an immune complex, such as hPD-1 •hPD-L1 .
  • immune (checkpoint) complexes form in trans between receptor-expressing T cells and ligandexpressing antigen-presenting cells (immune cells or cancer cells), creating a milieu known as the “immunological synapse”.
  • the inhibitory or stimulatory checkpoint receptors bind with their ligands, they mediate an immunosuppressive or immunoactivating signal to T cells, respectively.
  • T cells 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 progression 8 . Consequently, blockade of inhibitory immune checkpoints, e.g., through hPD-1 and/or hPD-L1 inhibition, has been proven effective in the treatment of several cancer types, including melanoma, non-small cell lung carcinoma, colorectal cancer, and hepatocellular carcinoma 9 . Interestingly, a considerable number of patients with no previous history of autoimmunity, who undergo treatment with such blockers, develop autoimmune side effects 10 . This observation aligns with a growing recognition of the significance of immune receptors in the pathogenesis of autoimmunity.
  • 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 serendipitous 2427 .
  • PPI interfaces tend to be flat. Consequently, classic medicinal chemistry methods are less effective for designing and identifying PPIs modulators 26 . Thus, rational and systematic approaches are necessary to guide the development of new PPI stabilizers.
  • 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.
  • Nbs NanobodiesTM
  • SOS1 »RAS complex were generated 28 utilizing the “Cross-linked PPIs and immunize llamas (ChILL)” and “Display and co-selection (DisCO)” methods as disclosed in WQ2016/012363.
  • ChoILL Cross-linked PPIs and immunize llamas
  • DisCO Display and co-selection
  • the present invention offers a solution to the aforementioned problems by introducing novel positive allosteric modulators (PAMs) of the human PD-1 »PD-L1 (hPD-1 »hPD-L1 ) complex.
  • PAMs positive allosteric modulators
  • the allosteric modulators disclosed herein improve the binding affinity within the hPD-1 »hPD-L1 complex through positive modulation of cooperative binding, wherein such cooperative binding is characterized by a cooperativity factor (a) greater than 1 .
  • the modulators By supporting the activity of the endogenous hPD-L1 ligand (orthosteric agonist), the modulators potentiate and/or prolong hPD-1-mediated immunosuppressive signaling, offering a targeted and effective approach to treating inflammatory conditions, including autoimmune diseases.
  • the allosteric modulators described herein exhibit specificity for epitopes predominantly present on the hPD-1 »hPD-L1 complex. More precisely, these modulators form ternary assemblies with the naturally occurring and transient hPD-1 »hPD-L1 complex. As a result, their activity is restricted to sites where both hPD-1 and hPD-L1 are present simultaneously- forexample, atthe immunological synapse where T cell activation occurs and/or at the site of inflammation. This contrasts with classical hPD-1 agonists, which activate hPD-1 systemically wherever the receptor is present, regardless of ligand engagement.
  • the modulators of the present invention are advantageous in that they enhance the natural hPD-1-mediated 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 PAMs may also allow for higher and more frequent dosing compared to conventional drugs, ensuring continuous saturation and improved therapeutic efficacy.
  • the PAMs of this invention may be useful for the treatment of inflammatory diseases, including autoimmune diseases, graft-versus-host disease, or allergies.
  • 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. DESCRIPTION OF THE FIGURES
  • Figure 1 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 ac t.
  • 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 receptor with an equilibrium dissociation constant K N b.
  • the model includes four key cooperativity factors: a: the factor of binding cooperativity between the orthosteric agonist L and the allosteric modulator Nb.
  • 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*).
  • R inactive conformation
  • R* signaling- competent active conformation
  • Immunizing mice with the receptor alone (left) leads to the maturation of conventional monoclonal antibodies that bind to the inactive conformation, thereby inhibiting the receptor.
  • 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 activestate conformation (R*).
  • a the factor of binding cooperativity between the orthosteric ligand (e.g., hPD-L1 ) and the allosteric nanobody (Nb);
  • p 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.
  • FIG. 3 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 PageRulerTM prestained Protein Ladder was used as a molecular weight marker.
  • Figure 4 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).
  • Figure 5 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 according to Example 4.
  • Figure 6 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).
  • G6, G11 contained sequence variants of CA19281 (SEQ ID NO: Figure 7.
  • 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.
  • 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.
  • FIG. 9 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.
  • FACS fluorescence-activated cell sorting
  • FIG. 10 Crystal structure of CA19279 in a complex with hPD-1.
  • CA19279 SEQ ID NO: 14
  • 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 .
  • Nanobodies that act as affinity modulators of the hPD-1 •hPD-L1 interaction do not modulate the hPD-1»hPD-L2 complex.
  • 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.
  • hPD-1 -Fc 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.
  • FIG. 12 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.
  • 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.
  • A Nanobody 102C12 and pembrolizumab are known antagonists of the hPD-1 pathway.
  • Rosnilimab and peresolimab are known agonists of the PD-1 pathway.
  • 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.
  • 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.
  • 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.
  • FIG. 16 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).
  • the Kabat numbering system was applied.
  • FIG. 17 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).
  • the Kabat numbering system was applied.
  • 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).
  • 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.
  • 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 20 Sequence alignment.
  • 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.
  • 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).
  • 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.
  • 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 notexpress PD-L1 , confirming that the functional effect of these constructs requires ligand engagement.
  • Figure 23 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 24 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.
  • FIG. 25 Cytokine release in a mixed lymphocyte reaction (MLR).
  • MLR mixed lymphocyte reaction
  • 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.
  • FIG. 26 Cytokine release in a mixed lymphocyte reaction (MLR).
  • MLR mixed lymphocyte reaction
  • 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 27 Characterization of epitopes via epitope binning using BLI.
  • Figure 28 Deep sequencing analysis of sequence-optimized variants A00247-A00253, derived from the same clonal cluster.
  • CDRs complementarity-determining regions
  • 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 29 Sequence alignment and CDR similarity analysis of hPD-1»hPD-L1 PAMs of the same clonal cluster.
  • Amino acid sequences and their alignment of molecules (SEQ ID NO: 35), A00113 (SEQ ID NO: 36), A00114 (SEQ ID NO: 37), A00115 (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).
  • Figure 30 Epitope mapping of the hPD-1»hPD-L1 complex PAM Nbs through screening of a PD- 1 mutant library.
  • 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 CA1881 1 and CA19275, it was the opposite for E61 K and S62Kwith CA19279 having higher binding strength.
  • 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.
  • PD-1 PD1 , PD1 , hPD-1 ” and “hPD1 ” may be used interchangeably and all refer to the same molecule, i.e., “human programmed cell death protein 1 ”.
  • PD-L1 PDL1 , hPD-L1 ” and “hPDL1 ” may be used interchangeably and all refer to the same molecule, i.e., “human programmed death-ligand 1”.
  • 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.
  • 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.
  • 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
  • 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.
  • affibody molecules 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,
  • 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).
  • 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.
  • 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.
  • 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 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.
  • 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 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.
  • 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.
  • 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.
  • 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 2B); and/or (iii) direct receptor stimulation that triggers downstream signaling, an effect possibly quantified by the allosteric factor y (see Figure 2B).
  • the direction and magnitude of these effects are not restricted for any given receptor;
  • Allosteric modulation of proteins 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) model 35 , 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) model 36 , 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.
  • MWC Monod-Wyman-Changeaux
  • KNF Koshland-Nemethy-Filmer
  • 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 switches 40 . When an allosteric modulator binds to a protein, it alters the free energy landscape within the conformational space of that protein.
  • Allosteric binding sites or epitopes 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 binders 42 .
  • 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 below 43 .
  • an allosteric modulator may exhibit selectivity either for a single molecular entity or for a multi-component assembly such as a complex, aggregate, or molecular chain.
  • an allosteric modulator targeting the hPD- 1 »hPD-L1 complex 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) hPD-1 alone, ii) hPD-L1 alone, oriii) both hPD-1 and hPD- L1 simultaneously.
  • 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 .
  • PAMs positive allosteric modulators
  • PAM positive allosteric modulator
  • 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.
  • 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 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.
  • 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 substrate 45 .
  • 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 ligands 46 .
  • the binding of an allosteric agent influences the structural and/or functional state of the target.
  • cooperativity is a special case of allostery associated with ligand-induced conformational dynamics, which may provide free energy of allosteric coupling via entropic effects 47 .
  • 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.
  • 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.
  • 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.
  • 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 concentration 48 .
  • Homotropic cooperativity is the primary mechanism by which evolution steepens the binding curves of biomolecular receptors to produce more responsive input-output behavior 48 . Consequently, a significant portion of biological processes are regulated by oligomeric ligandbinding proteins, such as hemoglobin and the p97 ATPase 50 .
  • 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.
  • 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 ligands 51 .
  • 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).
  • 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 motions 52 . 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 nature 53 .
  • 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.
  • ITC isothermal titration calorimetry
  • Such interactions are driven by favorable changes in free energy, which can involve an increase in entropy and/or a decrease in enthalpy 55 .
  • 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.
  • 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.
  • the cooperativity factor a can be defined 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.
  • K d equilibrium dissociation constant
  • the effect of the allosteric modulator of the PD-1 »PD-L1 complex can be calculated 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.
  • the ratio i.e. factor a quantifies how the presence of the allosteric modulator affects the binding affinity between PD-1 and PD-L1.
  • factor a can be generalized to account for the combined interactions among multiple binding sites.
  • a may be calculated by comparing the association constants of the complex formation in the presence and absence of other ligands.
  • positive cooperativity 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.
  • 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.
  • 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.
  • negative cooperativity 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.
  • the binding sites interact in a manner that decreases the overall stability of the complex as more ligands bind.
  • 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.
  • positive cooperativity increases and negative cooperativity (a ⁇ 1 ) decreases the thermodynamic stability of the ternary complex 57 .
  • 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.
  • 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.
  • 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).
  • peptide polypeptide
  • amino acid chain or any otherterm 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.
  • protein is also intended to encompass products resulting from post-translational modifications of a polypeptide.
  • 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-linked glyco
  • 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.
  • SPPS solid-phase peptide synthesis
  • thioester-forming ligation thioester-forming ligation
  • oxime and hydrazone-forming ligation thiazolidine/oxazolidine-forming ligation
  • ligation by disulfide exchange thioacid-capture ligation
  • 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 hist
  • 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.
  • 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.
  • 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 it 58 .
  • 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 tyrosine-based 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.
  • 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 (lymphocyteactivation gene 3), TIM-3 (T-cell immunoglobulin and mucin-domain containing-3), and TIGIT (T cell immunoreceptor with Ig and ITIM domains).
  • the term “receptor” specifically refers to hPD-1 , unless specified otherwise.
  • 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.
  • 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.
  • 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.
  • immune cells encompass, without limitation, all mature and immature forms of: i) lymphoid cells, including T cells (comprising helper, cytotoxic, and regulatory? 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 invariant?
  • MAIT invariant natural killerT
  • iNKT invariant natural killerT
  • myeloid cells including macrophages, dendritic cells, neutrophils, monocytes, mast cells, eosinophils, and basophils.
  • immune cell refers to both naturally occurring and engineered/non-natu rally occurring immune cells, the latter group including?
  • CAR-T 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) cells 61 , cytokine-induced killer (CIK) cells 62 , chimeric antigen receptor-modified macrophages (CAR-M cells) 63 , and CXCL9/10-engineered dendritic cells 64 , among others.
  • CAR-T cells chimeric antigen receptors
  • TCR-T cells T cells expressing an engineered T cell receptor
  • TCR-NK cells TCR-modified NK cells
  • CIK cytokine-induced killer
  • CAR-M cells chimeric antigen receptor-modified macrophages
  • CXCL9/10-engineered dendritic cells 64 among others.
  • ligand 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.
  • 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.
  • a ligand that binds to a receptor without activating the physiological response is termed a “receptor antagonist”.
  • the term “ligand” encompasses naturally occurring/endogenous, recombinant, and synthetic ligands.
  • 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.
  • receptor-ligand interactions 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.
  • 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 molecules 65 .
  • the complementarity between the surfaces of the binding proteins relies on both the shape/configu ration 66 and electrostatic properties of the binding sites 6768 .
  • the kinetics of receptorligand binding is characterized by parameters encompassing the “binding affinity” and “kinetic rates”, as described here.
  • 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 fluctuations 71 .
  • these energy contributions are intricately linked to the protonation states and cannot be calculated independently of such states 65 .
  • ligand of an immune receptor 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.
  • PD-1 receptor expressed on T cells its ligands PD-L1 and PD-L2 are typically membrane-bound proteins found on antigen-presenting cells (APCs) or other immune cells.
  • APCs antigen-presenting cells
  • PD-1 interacts with these ligands, it attenuates T cell activation, thereby regulating immune responses and maintaining peripheral tolerance.
  • 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.
  • 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.
  • 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.
  • affinity refers to the strength of a reversible interaction between at least two molecules, such as a ligand and a receptor.
  • a high-affinity interaction elicits a robust biological response
  • a weak-affinity interaction induces a minimal biological response 72 .
  • a simple reversible reaction between ligand L and receptor R can be
  • the saturation binding curves may adopt a sigmoidal shape 73 .
  • 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.
  • 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:
  • 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 constant 74 .
  • 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.
  • [R t ] and [M t ] are total concentrations of the receptor and allosteric modulator, respectively.
  • thermodynamics 75 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 forces 76 .
  • G Gibbs free energy
  • AG Gibbs free energy
  • AG AG° + RT InQ.
  • 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.
  • 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.
  • a positive AG indicates a non-spontaneous process (unfavorable interaction), whereby external energy input is required to form or maintain a complex.
  • 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).
  • 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.
  • AG a 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.
  • 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.
  • 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.
  • the heat effect of a binding reaction is a global property, encompassing contributions from both the solute and the solvent 78 .
  • 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 contributions 79 .
  • ASsdv accounts for solvent entropy changes due to surface burial upon binding, often beneficial due to its typically positive value;
  • AS con f 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 complex 81 ;
  • 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 .
  • 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 AG 84 .
  • 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 complex 86 .
  • 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 binding 55 .
  • 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.
  • 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.
  • 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 target 75 .
  • 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.
  • 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.
  • the allosteric modulator of the PD-1 »PD-L1 complex 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 surface 87 .
  • Such antigenic surfaces of immune receptorligand complexes enable specific binding of the allosteric modulators disclosed in this application.
  • 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.
  • 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 designates a cluster of molecules comprising at least one protein, stabilized by 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.
  • 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 “protein-protein complex”.
  • a protein complex is formed by a receptor and a ligand, or a plurality of these.
  • 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.
  • assemblies of protein complexes can result in homo-multimeric or heteromultimeric complexes, characterized by interactions that range from transient to stable.
  • multiprotein complexes form by stochastic interactions between mature proteins, via chaperone- facilitated interactions, or through co-translational mechanisms 88 .
  • 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.
  • 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.
  • 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.
  • 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.
  • polar amino acids 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.
  • posttranslational and other modifications of a polypeptide chain can influence the 3D structure of a protein.
  • ligand binding including the binding of allosteric modulators
  • phosphorylation e.g., phosphorylation
  • sulfation e.g., phosphorylation
  • glycosylation e.g., glycosylation
  • hydrophobic groups e.g., hydrophobic groups
  • the interacting partners induce reciprocal conformational changes duringthe binding process that are necessary for specific recognition and binding, as described earlier.
  • all molecular recognition processes require some degree of conformational adaptation 89 .
  • 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 chains 90 .
  • 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.
  • conformational entropy 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.
  • 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 coefficients 39 .
  • formation 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.
  • 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.
  • 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.
  • induce refers to causing, triggering, enhancing, or bringing about a specific effect, action or result, through direct or indirect means.
  • 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.
  • murine models deficient in specific inhibitory receptors exhibit a heightened susceptibility to autoimmune diseases and increased immunopathology during infection 93 .
  • 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 autoreactivity 11 ’ 93 .
  • the human genome encodes more than 300 genes encoding potential immune-inhibitory receptors 94 . 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 function 95 .
  • ITSM immunofluoreceptor tyrosine-based switch motif
  • SH2 domain-containing protein tyrosine phosphatase 1 SHP-1
  • Src homology region 2 domain-containing phosphatase-2 SHP-2
  • SH2- containing inositol phosphatase-1 SHIP
  • CSK inhibitory C-terminal Src kinase
  • 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.
  • 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 signaling 102 .
  • inhibitory receptors function not as a binary on/off switches but rather as a “volume control” systems 103 .
  • 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 specificity 103 .
  • 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.
  • Poliovirus receptor also known as CD155
  • PVR is a negative feedback receptor upregulated on dendritic cells following TLR stimulation or upon CD4OCD40L complex formation 106 .
  • 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.
  • PD-1 expression is induced upon activation of naive T cells, where it acts as a negative feedback receptor 107 .
  • PD-1 is also highly expressed on effector T cells 108 , where it functions as a threshold receptor.
  • PD-1 is involved in mediating ? cell exhaustion 109 . 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.
  • PD-L1 PD-L1
  • IFNy IFNy
  • PD-L1 negative feedback ligand
  • PD- 1 »PD-L1 signaling has characteristics of both a threshold and a negative feedback regulatory axis.
  • CTLA-4 acts as a negative feedback receptor on conventional ? cells, which upregulate its expression upon activation 110 .
  • C?LA-4 is constitutively expressed on regulatory ? cells, where it may operate as a threshold receptor.
  • 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.
  • LAIR-1 is expressed in resting ? cells, where it serves as a threshold receptor 111 .
  • 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 feedback 103 .
  • immune checkpoint inhibition refers to a therapeutic strategy or intervention that targets one or more immune checkpoint proteins to modify the regulation of an immune response. ?he targeting involves counteractingthe function, disruptingthe structure and/or modifying the localization of at least one immune checkpoint protein. ?his 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.
  • ?hese agents are well-known in the art and include, without limitation: Pembrolizumab (Keytruda®), which targets PD-1 ; ?remelimumab (Imjudo®), which targets C?LA-4; Durvalumab (Imfinzi®), which targets the PD-1 »PD-L1 interaction; Nivolumab (Opdivo®), which targets PD-1 ; and Relatlimab, which targets LAG-3.
  • ?hese 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.
  • modulate refers to 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).
  • baseline functioning or activity refers to the default signaling of the PD-1 »PD-L1 complex, in the absence of any external (allosteric) modulator affecting such signaling.
  • the signaling activity may be characterized by its strength, duration, and other features that could be subject to modulation.
  • modulation of a protein complex may occurthrough: 1 ) binding of an agent to 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.
  • Various embodiments described herein refer to the “allosteric modulation” of the hPD-1 »hPD-L1 complex, wherein the allosteric modulation occurs through the binding of an allosteric modulator to the protein complex, which induces conformational changes within the complex. These conformational changes lead to the induction of positive cooperativity within the complex, resulting in an increased affinity between the constituents of the complex. This enhanced affinity subsequently causes prolonged or potentiated immunosuppressive signaling mediated by the PD- 1 »PD-L1 interaction. Through this mechanism, the allosteric modulator stabilizes and reinforces the naturally occurring inhibitory immune checkpoint, enabling a targeted and spatiotemporally selective modulation of immune responses.
  • antibody refers to an immunoglobulin (Ig) molecule or a molecule containing an Ig domain, which specifically binds with an antigen, including multimers thereof.
  • Ig immunoglobulin
  • 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.
  • V variable region
  • F(ab) fragment antigen-binding region
  • 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.
  • 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).
  • CL constant domain
  • VL variable domain
  • 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 refers 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.
  • CDRs complementarity-determining-regions
  • 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 (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.
  • 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.
  • CDRs complementary metal-oxide-semicon determining regions
  • 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.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • CDRs complementarity determining regions
  • the antigen-binding domain of a conventional 4-chain antibody e.g., IgG, IgM, IgA, IgD, or IgE, as commonly understood
  • a conventional 4-chain antibody e.g., IgG, IgM, IgA, IgD, or IgE, as commonly understood
  • 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
  • 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
  • immunoglobulin single variable domain 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.
  • 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.
  • the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDR’s.
  • 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.
  • a light chain variable domain sequence e.g., a VL sequence
  • a heavy chain variable domain sequence e.g., a VH or VHH sequence
  • the immunoglobulin single variable domain may be a Nanobody” (also referred to as “Nanobody”, “nanobody” or “Nb”), or a suitable fragment thereof.
  • Nanobody also referred to as “Nanobody”, “nanobody” or “Nb”
  • Nanobody Nanobodies
  • Nanoclone are registered trademarks of Ablynx N.V. (a Sanofi Company).
  • WG2008/020079 for a general description of nanobodies, reference is made to the detailed explanation below and the prior art cited herein, such as to WG2008/020079.
  • 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 .
  • Ig antigen binding immunoglobulin
  • HCAbs heavy chain antibodies
  • 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., 7997 113 ).
  • VHH domain variable domain of the light chain
  • CDR3 complementarity-determining region 3
  • 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 titre and a complex repertoire of HCAbs can be obtained from immunized or infected dromedaries or llamas 114 .
  • 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").
  • VHHs and nanobodies 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, WC95/04079 and WO96/34103 of the Vrije Universiteit Brussel; WO94/25591 , WO99/37681 , WC00/40968, WC00/43507, WC00/65057, WC01 /40310, WC01 /44301 , EP1134231 and WO2/48193 of Unilever; WO97/49805, WC01 /21817, WC03/035694, WC03/054016 and WC03/055527 of the Vlaams Instituut voor Biotech nologie (VI B); WC03/050531 of Algonomics N.V.
  • a nanobody/Nb in particular VHH sequences and partially humanized nanobodies
  • 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.
  • CDR3 complementarity-determining region 3
  • 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., 7996 117 .
  • 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.html 118 ), Chothia 119 , Kabat 113,120 , or IMGT 121 .
  • CDRs complementarity-determining regions
  • FRs framework regions
  • 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.
  • 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 maturation 122 . 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.
  • 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 sequence 123 .
  • 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.
  • 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.
  • other suitable humanizing substitutions or suitable combinations thereof can be determined by the skilled person.
  • 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 may have several advantages, such as a reduced immunogenicity, compared to the corresponding naturally occurring VHH domains.
  • “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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • these antibody fragments or single domain antibodies 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.
  • Fc-fusion constructs may include one or more VHHs or nanobodies, as described herein, and may combine the antigen- binding specificity of the single domain antibodies with the effector functions and extended halflife conferred by the Fc domain.
  • biopanning refers to an affinity selection technique used to identify molecules that bind to a specific target.
  • 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.
  • medicaments 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.
  • oral medicaments 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.
  • 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).
  • 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 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.
  • 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.
  • treating and its variations may encompass preventive or prophylactic measures against an injury, disease, or pathological condition.
  • treatment 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 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.
  • communicable diseases examples include influenza, tuberculosis, AIDS, malaria, and COVID-19.
  • 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 refers 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.
  • p53 has been described as an autoantigen in several autoimmune diseases, including lupus and scleroderma
  • TPO thyroid peroxidase
  • GAD glutamic acid decarboxylase
  • 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 cells 124 .
  • self-reactive T cells particularly CD4 + T cells, are implicated in mediating various aspects of autoimmune inflammation 125 ; in some diseases, they may function as effector cells directly involved in the killing of cells expressing their target autoantigens.
  • 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 tissues 126 .
  • HLA human leukocyte antigen locus 127 .
  • APCs antigen-presenting cells
  • DCs dendritic cells
  • 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 .
  • MHC major histocompatibility complex
  • 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)antigens 130 .
  • 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 conditions 131 .
  • 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 cells 130 .
  • co-stimulatory and co- inhibitory receptors and their ligands also play a role in regulating immune responses of natural killer (NK) cells, innate lymphoid cells, and myeloid cells 132 .
  • NK natural killer
  • Autoreactivity associated with autoimmune conditions 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?
  • 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 men 133 .
  • the clinical presentations of autoimmunity are markedly diverse; the manifestations span from subtle abnormalities that evade diagnosis/detection to acute, potentially life-threatening organ dysfunction 3 .
  • autoreactivity exists on a spectrum, ranging from a basal physiological level essential for lymphocyte selection and the maintenance of immune system homeostasis 5 , 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 damage 134 .
  • 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.
  • 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 vitiligo 1 .
  • the list of autoimmune diseases provided above is not exhaustive.
  • graft versus host disease refers to a complex immunological disorder that occurs when immunocompetent donor-derived immune cells, primarily? 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 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.
  • AR allergic rhinitis
  • AAS allergic asthma syndrome
  • AD atopic dermatitis
  • FA food allergy
  • 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.
  • inflammatory cells e.g., mast cells, CD4 + T cells, B cells, macrophages, and eosinophils
  • Th2 cells have been associated with the production of immunoglobulin E (IgE) and cytokines like IL-3, IL-4, IL-5, and IL- 13.
  • IgE immunoglobulin E
  • cytokines like IL-3, IL-4, IL-5, and IL- 13.
  • Tfh follicular helper T
  • 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.
  • 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.
  • 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.
  • 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 hist
  • 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.
  • 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 deoxyribonucleic acid
  • RNA ribonucleic acid
  • 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).
  • 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 refers 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.
  • viral vectors including lentiviral and adenoviral vectors
  • 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.
  • 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.
  • gene therapy 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.
  • 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.
  • 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.
  • a pharmaceutical or medicinal composition refers to any formulation comprising an active ingredient - such as the ISVD- comprising allosteric modulator of the hPD-1 »hPD-L1 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.
  • an active ingredient - such as the ISVD- comprising allosteric modulator of the hPD-1 »hPD-L1 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.
  • 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.
  • 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.
  • carrier refers 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.
  • 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.
  • excipients examples include 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 orvolume. 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.
  • 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.
  • pharmaceutically acceptable carriers 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.
  • 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.
  • parenteral e.g., intravenous, intramuscular, subcutaneous, or intradermal
  • topical transdermal
  • nasal buccal
  • sublingual sublingual
  • pulmonary e.g., via inhalation or aerosol
  • ophthalmic otic, rectal, vaginal, urethral
  • intraperitoneal intracardiac, in
  • 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.
  • compositions described herein 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 the hPD-1 »hPD-L1 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
  • the present invention originates from research endeavors directed toward developing novel and precise strategies for modulating human PD-1 (hPD-1 )-mediated signaling pathways, which play a central role in immune regulation.
  • the invention relates to allosteric modulators that bind to one or more conformational epitopes located on the hPD-1 »hPD-L1 complex, and, upon binding, induce positive cooperativity within this complex.
  • the unique ability of the allosteric modulators to enhance cooperative interactions within the hPD-1 »hPD-L1 complex represents the central concept of the present disclosure.
  • the modulators described herein are designed to reinforce and stabilize the natural interaction between hPD-1 and its ligand hPD-L1 . This results in a controlled amplification of the downstream inhibitory signaling cascade.
  • modulation occurs in a (i) targeted fashion, i.e., restricted to immune synapses where the complex is formed; (ii) spatiotemporally selective manner, i.e., limited to the time and location of complex formation; and (iii) hPD-L1 (ligand)-dependent mode.
  • targeted fashion i.e., restricted to immune synapses where the complex is formed
  • spatiotemporally selective manner i.e., limited to the time and location of complex formation
  • hPD-L1 (ligand)-dependent mode Collectively, these features confer a high degree of specificity and therapeutic control.
  • the invention provides an innovative therapeutic approach for conditions involving aberrant or excessive immune activation, including autoimmune diseases, graft-versus-host disease (GVHD) and allergic diseases.
  • GVHD graft-versus-host disease
  • the invention provides novel modulators that are capable of allosterically binding to epitopes located on the hPD-1 »hPD-L1 complex.
  • Said modulators comprise an immunoglobulin single variable domain (ISVD), as defined herewith in detail, with an antigen-bindingsite specifically targeting the hPD-1 »hPD-L1 complex.
  • ISVD immunoglobulin single variable domain
  • the hPD-1 »hPD-L1 complex Upon binding to the allosteric modulator disclosed herein, the hPD-1 »hPD-L1 complex exhibits increased affinity between its interacting subunits, that is between the receptor (hPD-1 ) and the ligand (hPD-L1), as defined herein.
  • 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 in this application.
  • the present disclosure applies the cooperativity framework to describe the interdependent behavior of binding sites within the hPD- 1 »hPD-L1 complex upon interaction with the allosteric modulator disclosed herein.
  • the ISVD of the allosteric modulator of this invention specifically binds to an epitope that is predominantly present on the hPD-1 »hPD-L1 complex. This means that the epitope may not be exclusively present on this complex, although it can be exclusively present on said complex.
  • the epitope to which the allosteric modulator disclosed herein binds may be: (i) present exclusively on hPD-1 engaged in a complex with hPD-L1 ; or (ii) present exclusively on hPD-L1 engaged in a complex with hPD-1 ; or (iii) formed by residues contributed jointly by both hPD-1 and hPD-L1 engaged in the complex.
  • the epitope may correspond to a conformational determinant that only arises upon complexation of hPD-1 and hPD-L1 , such that it is absent or structurally distinct in the unbound (monomeric) forms of the individual proteins, that is unbound hPD-1 and unbound hPD- L1.
  • the epitope may partially overlap with amino acid residues and/or structural features present in the uncomplexed receptor (hPD-1 ) and/or ligand (hPD-L1), 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.
  • 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 PD-1 and/or PD-L1 .
  • 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 hPD- 1 »hPD-L1 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.
  • 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 (hPD-1 ) or ligand (hPD-L1). 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 hPD-1 »hPD-L1 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.
  • the cryptic epitope may correspond to a loop region or flexible domain in hPD-1 that adopts a defined conformation only upon hPD-L1 engagement.
  • a binding pocket or groove formed at the hPD-1 »hPD-L1 interface may be absent in either protomer alone, but is formed or stabilized upon complex formation.
  • 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 hPD-1 »hPD-L1 complex ratherthan with the unbound hPD-1 or hPD-L1 protomers.
  • the invention provides allosteric modulators that specifically target the transient human (hPD-1 »hPD-L1 complex.
  • transient complex and “transient protein-protein interaction” refer to a non-permanent, reversible interaction between hPD-1 and hPD-L1.
  • transient complexes are capable of dynamically associating and dissociating under physiological conditions, and each constituent protein, that is hPD-1 or hPD-L1 , 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.
  • hPD-1 is capable of forming transient complexes with both of its ligands, PD-L1 and PD-L2, during the 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.
  • TCR T cell receptor
  • the hPD-1 »hPD-L1 and hPD-1 »hPD- L2 interactions are dynamically regulated and are reversible, enabling precise control of T cell activity during physiological and pathological immune events.
  • the allosteric modulators disclosed herein are specifically raised to bind to and modulate the hPD-1 »hPD-L1 complex, and not the hPD-1 »hPD-L2 complex.
  • the immunoinhibitory complex of hPD-1 »hPD-L1 bound by the allosteric modulator of the present invention comprises one or more subunits derived from a human (Homo sapiens).
  • said subunits include naturally occurring isoforms, polymorphic variants, mutants, or engineered derivatives thereof, as discussed in detail below.
  • 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.
  • 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.
  • 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 hPD-1 or hPD-L1 , as determined by standard sequence alignment algorithms (e.g., BLAST, ClustalW, or equivalent).
  • 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 the hPD- 1 »hPD-L1 complex targeted/bound 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 encompassed herewith.
  • the hPD-1 »hPD-L1 complex 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).
  • the hPD-1 »hPD-L1 complex may comprise a membrane-bound hPD-L1 isoform or a soluble hPD-L1 isoform.
  • the hPD-1 »hPD-L1 complex may include functionally active mutants, gain- or loss- of-function variants, or rare alleles associated with pathological or protective phenotypes.
  • the invention encompasses allosteric modulators that target and/or bind to the hPD- 1 »hPD-L1 complex, whether composed of wild-type proteins, mutants and/or engineered variants, provided that such complexes are capable of presenting a modulator-specific epitope suitable for selective allosteric binding, as described herein.
  • the allosteric modulator disclosed herein selectively binds to the hPD- 1 »hPD-L1 complex, wherein hPD-1 is localized on the surface of an immune cell.
  • the immune cell may be either a mature (fullydifferentiated) or immature (not fully differentiated) cell of the immune system.
  • the immune cell is of lymphoid lineage.
  • Non-limiting examples of such cells include: (i) T lymphocytes (T cells), including but not limited to helper T cells (Th), cytotoxic T cells (Tc), and regulatory T 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 invariant T (MAIT) cells and invariant NKT (iNKT) cells.
  • the immune cell is of myeloid lineage.
  • Nonlimiting examples of such cells include macrophages, dendritic cells, neutrophils, monocytes, mast cells, eosinophils, and basophils.
  • the allosteric modulator of the present invention which specifically binds to an epitope predominantly present on the hPD-1 »hPD-L1 complex, 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 hPD-L1 to hPD-1 (or vice versa), thereby impacting the complex stability.
  • an a value greater than 1 indicates that binding of the allosteric modulator enhances the bindingof hPD-1 to hPD-L1 , hence demonstrating positive allosteric modulation of the hPD-1 »hPD- L1 complex.
  • the allosteric modulator of the present invention which specifically binds to the hPD-1 »hPD-L1 complex, enhances the binding affinity of hPD-L1 to hPD-1 within said complex.
  • This enhancement is quantitatively characterized by a cooperativity factor (a) greater than 1 , as measured for the hPD-1 »hPD-L1 interaction in the presence of the allosteric modulator.
  • a cooperativity factor a ternary complex is formed comprising the immune receptor (hPD-1 ), its ligand (hPD-L1), and the allosteric modulator.
  • the cooperativity factor a is defined as the ratio of the equilibrium dissociation constant (K D ) for the binary receptor-ligand complex (hPD-1 »hPD- L1 ) to the equilibrium dissociation constant (K D ) for the ternary complex (hPD-1 »hPD- L1 •modulator).
  • K D equilibrium dissociation constant
  • K D ternary complex
  • An a value greater than 1 indicates that the presence of the allosteric modulator increases the affinity between hPD-1 and hPD-L1 , thereby demonstrating positive heterotropic cooperativity, as defined herein.
  • the invention provides an allosteric modulator that enhances immunosuppressive signalingof the hPD-1 »hPD-L1 complex.
  • 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 hPD-1 in complex with hPD-L1 , thereby enhancing/inducing/promoting the activity of downstream inhibitory signaling cascades.
  • the modulation of downstream signaling by the allosteric modulator disclosed herein is achieved through enhanced affinity between hPD-1 and 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 the hPD- 1 »hPD-L1 complex, as measured by factor 6 (delta) greater than 1 .
  • Factor 6 reflects the influence of the allosteric modulator - ligand (hPD-L1) interaction on the functional activation state of the receptor (hPD-1), independent of binding affinity alone.
  • the allosteric modulator disclosed herein may function as an efficacy modulator, capable of inducing a conformational change within the receptor engaged in the target complex (hPD-1 •hPD-L1 ) that is transmitted to intracellular domains involved in signal transduction, thereby modulating the receptor’s downstream signaling capacity.
  • the allosteric modulator of this disclosure functions as a positive allosteric modulator (PAM) that enhances the effects of a ligand (hPD-L1) bound to the orthosteric site of hPD-1.
  • 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.
  • PAMs may also improve receptor efficacy, which refers to the magnitude of the response triggered by activation through the orthosteric ligand (factor delta).
  • the PAM of this invention stabilizes the hPD-1 »hPD-L1 complex and promotes immunosuppressive signaling.
  • PD-1 Programmed cell death-1
  • 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 ? 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.
  • IgV extracellular immunoglobulin variable
  • 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).
  • ITIM immunoreceptor tyrosinebased inhibitory motif
  • ITSM immunoreceptor tyrosine-based switch motif
  • 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.
  • 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 modulators disclosed in this application modulate downstream signaling to promote immunosuppressive pathways. As such, they hold strong therapeutic potential for conditions characterized by excessive or dysregulated inflammatory responses, including autoimmune diseases, allergy, or GVHD. These modulators preserve the native spatial and temporal dynamics of immune signaling, supporting receptor activation mediated by endogenously formed ligand-receptor interactions.
  • 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/organs.
  • APCs antigen-presenting cells
  • the allosteric modulator of this invention exhibits spatiotemporal selectivity for the hPD-1 »hPD-L1 complex.
  • spatialotemporal selectivity is meant that the modulator acts specifically on the naturally formed hPD-1 »hPD-L1 complex, thereby rendering its modulatory activity dependent on the presence of hPD-L1 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 hPD-1 »hPD-L1 interaction naturally occurs. This spatiotemporal selectivity ensures that the allosteric modulator of this invention exerts its effects only in tissues and/or microenvironments where hPD-1 and hPD-L1 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.
  • the modulator 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 hPD-1 and hPD-L1 are engaged in a complex.
  • the allosteric modulator of this disclosure binds to the hPD-1 »hPD-L1 complex 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 inhibition 140 .
  • 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.
  • APCs antigen-presenting cells
  • B cells that express corresponding ligands such as PD-L1 , B7-1 , B7-2, or HVEM.
  • NK natural killer
  • 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 142 .
  • the allosteric modulator described herein specifically binds to a conformational epitope present on the hPD-1 »hPD-L1 complex.
  • the allosteric modulator may recognize and bind to conformational epitopes located on: i) both subunits of the complex, meaning both the receptor a nd the ligand molecules involved in the complex, orii) a single subunit of the complex, i.e., either the receptor molecule (hPD-1) or the ligand molecule (hPD-L1) of the complex.
  • the allosteric modulator may bind to any number of hPD- 1 »hPD-L1 complex-associated epitopes. It shall be understood that the induction of positive cooperativity within hPD-1 »hPD-L1 by the allosteric modulator of this invention can be achieved irrespective of the number or combination of conformational epitopes recognized and bound by the modulator.
  • 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.
  • 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.
  • the allosteric modulator of this invention specifically binds to the hPD-1 »hPD-L1 complex through at least one or more, preferably 3 complementarity- determining region(s) (CDR(s)), wherein the CDR comprises any of the amino acid sequences as depicted in SEQ ID NOs: 76, 77, or 78.
  • CDR(s) 3 complementarity- determining region(s)
  • 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.
  • CDRs complementarity-determining regions
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the exemplified humanized variants allow for framework residue substitutions as typically known in the art.
  • 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.
  • the humanized variant 1 FR1 has a substitution of 5V
  • hypothetical humanized variant 2 FR1 has a substitution of 11 P
  • these combination of both substitutions is disclosed herein as a further alternative humanization.
  • hypothetical humanized variant 1 FR1 has a substitution of 5V and 1 D
  • 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.
  • the allosteric modulators of this invention are derived from the innate or adaptive immune system.
  • these binding agents are preferably derived from immunoglobulins, with antibodies or antibody fragments being particularly preferred.
  • 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).
  • a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen-binding site.
  • 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.
  • the binding agent of this disclosure comprises an immunoglobulin single variable domain, specifically a NanobodyTM (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.
  • any of the allosteric modulators of the 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.
  • 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.
  • PEG polyethylene glycol
  • XTEN polypeptides such as methoxypoly(ethyleneglycol) or mPEG
  • Fc domains such as 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 allosteric modulators of the hPD-1 »hPD-L1 complex may be modified and/or may comprise (or can be fused to) other moieties.
  • 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.
  • 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.
  • Such functional groups 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 A 143 .
  • 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.
  • 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).
  • PEG polyethylene glycol
  • Various forms of pegylation similar to those utilized in antibody and antibody fragment engineering (including single-domain antibodies and ScFv), can be applied 144-146 .
  • site-directed pegylation via a cysteine residue is employed 147 .
  • 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.
  • 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 hPD-1 »hPD-L1 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
  • 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.
  • 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).
  • DOTA 2,2',2"-(10-(2- ((2,5-dioxopyrrolidin-1 -yl)oxy)-2-oxoethyl)-1 ,4,7,10-
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a carrier including carriers suitable for pharmaceutical purposes.
  • One non-limiting example is the liposomal formulations described by Cao and Suresh, 2000' 48 .
  • Such binding pairs may also be used to link a therapeutically active agent to the binding agent of the invention.
  • linker molecules 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 proteins 149 .
  • linker sequences include (GS)5 (GSGSGSGSGS; SEQ ID NO: 23), (GS)10 (GSGSGSGSGSGSGSGSGSGSGSGS; 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).
  • the linker molecule comprises or consists of one or more particular sequence motifs.
  • 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 ).
  • 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' 50 .
  • 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.
  • the ISVD-comprising modulator of this invention 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.
  • the VHH or its variant may be fused to an Fc domain, particularly a human IgGI Fc tail, 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.
  • 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).
  • 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.
  • 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.
  • the ISVD-comprising modulator of this invention may be provided in the format of a knob- into-hole (KiH) fusion construct.
  • 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) 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.
  • 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.
  • the allosteric modulator of this invention comprises an ISVD comprising an amino acid sequence selected from SEQ ID NOs: 52, 60, 61 , 62, 63, 65, 67, 69, 70, 72, 73, 74, 75, or 185-239.
  • the allosteric modulator comprises an ISVD including any of the aforementioned sequences, wherein the sequence is further optimized and/or humanized, as described herein.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the allosteric modulator of this invention is used in the treatment of an autoimmune disease, as defined herein.
  • 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.
  • 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.
  • said allosteric modulator, nucleic acid, vector, or pharmaceutical composition can be used in the treatment of organ-specific autoimmune diseases.
  • organ-specific autoimmune 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.
  • the allosteric modulator, nucleic acid, vector, or pharmaceutical composition is used for treating inflammatory bowel diseases, including Crohn’s disease and ulcerative colitis.
  • the allosteric modulator, nucleic acid, vector, or pharmaceutical composition is employed in the treatment of autoimmune skin diseases.
  • diseases include, but are not limited to, psoriasis, vitiligo, pemphigus, pemphigoid, bullous pemphigoid, and epidermolysis bullosa acquisita.
  • 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.
  • CIDP chronic inflammatory demyelinating polyneuropathy
  • said modulator, nucleic acid, vector, or pharmaceutical composition is used in the treatment of autoimmune hematologic diseases.
  • autoimmune hemolytic anemia AIHA
  • immune thrombocytopenia ITP
  • acquired hemophilia cold agglutinin disease
  • paroxysmal nocturnal hemoglobinuria PNH
  • the allosteric modulator, nucleic acid, vector, or pharmaceutical composition can be used for the treatment of vasculitis and autoimmune rheumatic diseases.
  • 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.
  • 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.
  • autoimmune pancreatitis autoimmune polyendocrine syndromes (types 1 , 2, and 3)
  • autoimmune inner ear disease autoimmune retinopathy
  • autoimmune angioedema autoimmune angioedema
  • CRMO chronic recurrent multifocal osteomyelitis
  • 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, dermatomyo
  • the allosteric modulator of this invention may be used in the treatment of graft-versus-host disease (GVHD), as defined herewith.
  • GVHD graft-versus-host disease
  • the allosteric modulator of this invention may be used in the treatment of allergic diseases, as defined herewith.
  • 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.
  • this embodiment encompasses non-lgE- mediated hypersensitivity reactions, including contact dermatitis, allergic conjunctivitis, and certain forms of drug allergies.
  • 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.
  • the treatment includes treatment of occupational and environmental allergies triggered by allergens such as pollen, dust mites, molds, animal dander, and insect venoms.
  • kits which contain means to detect said protein complex, including the allosteric modulator or ISVDs 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 for in vivo imaging, or for diagnosis of an altered protein complex quantity, response or effect in a subject.
  • said kit 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.
  • the kits typically contain labelled or coupled protein-complex binding agents such as said allosteric modulator and/or ISVDs described herein.
  • the kits may contain labels for nucleic acids such as primers or probes.
  • kits 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.
  • 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.
  • the allosteric modulatoror bindingagent in particularcomprising a 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.
  • 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.
  • a detection agent such as a tag or a label.
  • 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.
  • detectable label or tag 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.
  • 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, biolum
  • 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 .
  • 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 labels will 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 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 imaging purposes, depending on the choice of the specific label.
  • an in vitro method 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 of tumor microenvironments, stromal compartments, or associated lymphoid tissues.
  • the ability to detect and spatially resolve the hPD- 1 »PD-L1 complex 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 PD-1 and or PD-L1 .
  • 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).
  • NSCLC non-small cell lung carcinoma
  • RCC renal cell carcinoma
  • HNSCC head and neck squamous cell carcinoma
  • HCC hepatocellular carcinoma
  • CRCC colorectal cancer
  • TNBC triple-negative breast cancer
  • various hematologic malignancies such as classical Hodgkin lymphoma (cHL), non-
  • 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.
  • IHC immunohistochemistry
  • IF immunofluorescence
  • PKA proximity ligation assay
  • detection of the hPD-1 »hPD-L1 complex, but not free PD-1 or PD-L1 can be enabled through the use of conformation-specific binders such as the allosteric modulators (PAMs) disclosed herein, which selectively recognize the receptor-ligand complex in its native state.
  • PAMs allosteric modulators
  • 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 complexpositive cells within tumor nests versus immune-infiltrated regions.
  • TMB tumor mutational burden
  • MSI microsatellite instability
  • gene expression signatures e.g., interferon-y response
  • 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.
  • 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.
  • 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.
  • said allosteric modulator wherein said positive cooperativity induction is determined by a factor a value higher than 1 .
  • 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.
  • 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.
  • 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).
  • 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.
  • said method wherein the positive cooperativity is determined by a factor a value higher than 1 .
  • 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.
  • 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).
  • ISVDs immunoglobulin single variable domains
  • Nbs nanobodies
  • 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.
  • said method involves displaying candidate modulators on a cell surface via display biopanning, including phage display, yeast display, or mammalian display.
  • Example 1 The cubic ternary complex model of allosteric modulation of receptor activation.
  • the (R) and (R*) states can be selectively targeted by (natural/endogenous) ligands (L) bindingtothe 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.
  • 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.
  • 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.
  • R* active state receptor
  • immune receptor-ligand complexes were stabilized using (bio)chemical cross-linking methods.
  • 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 2A).
  • allosteric binders i.e., allosteric Nanobodies
  • hPD-1 affinity for its orthosteric ligand hPD-L1
  • hPD-1 activity Figure 2B
  • Example 3 Immunization of a llama with a cross-linked hPD-1 •hPD-L1 complex to generate an antibody immune library.
  • 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 152 . Protein refolding, integrity, purity and functionality were evaluated by size exclusion chromatography, SDS- PAGE and biolayer interferometry (BLI) on Octet (data not shown).
  • glutaraldehyde was used as a chemical crosslinker in the concentration range from 0.29% to 0.04% ( Figure 3).
  • 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.
  • 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.
  • 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 vector 153 .
  • Example 4 Selection of allosteric Nanobodies that bind and stabilize the hPD-1 •hPD-L1 complex.
  • 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 4.
  • 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.
  • Round 2a we used immobilized hPD-1 in combination with soluble hPD- L1 .
  • 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.
  • 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.
  • hPD-1 -hFc AcroBiosystems, PD1 -H5257
  • hPD-L1-hFc AcroBiosystems, PD1 -H5258
  • 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 CaptureSelectTM 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 5). 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.
  • Example 6 Screening for Nanobodies that stabilize the non-cross-linked hPD-1*hPD-L1 complex by ELISA.
  • Example 3 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.
  • Nbs were subsequently biotinylated using a fivefold molar excess of EZ-LinkTM 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.
  • Nanobodies 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.
  • these same Nanobodies exhibit minimal to no binding to the individual protomers (Figure 7). 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 .
  • hPD-1 »hPD-L1 the separate protomers
  • hPD-1 alone or hPD-L1 alone the separate protomers
  • the cooperativity factors a were 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.
  • Nanobodies act as allosteric affinity modulators of the hPD-1 •hPD-L1 complex on Jurkat cells expressing hPD-1 .
  • Nanobodies act as affinity modulators of hPD-L1 for the hPD- 1 receptor expressed on human cells.
  • 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.
  • 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 startingfrom 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 (1x10 5 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.
  • 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 10A; Example 8).
  • CA19279 was selected based on its ability to bind to a complexspecific conformation, i.e., hPD-1 »hPD-L1 complex-specific conformation.
  • hPD-1 »hPD-L1 complex-specific conformation i.e., hPD-1 »hPD-L1 complex-specific conformation.
  • hPD-1 and CA19279 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 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 strands 155 .
  • hPD-L1 binds the “front” 0 sheet of hPD-1 (PDB:4ZQK).
  • 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) 156 . All intermolecular hydrogen bonds between CA19279 and hPD-1 are listed in Figure 10C.
  • the hPD-1 receptor is triggered by different natural ligands including hPD-L1 and hPD-L2.
  • 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
  • CA18811 SEQ ID NO: 12
  • CA19275 SEQ ID NO: 13
  • CA19281 SEQ ID NO: 15
  • an irrelevant Nb of SEQ ID NO: 16 targeting an irrelevant protein
  • hPD-1 hPD-1 -hFc, AcroBiosystems, PD1 -H5257
  • 10 ng of hPD-1 was immobilized on ELISA 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
  • 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.
  • 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.
  • 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).
  • 40 pL of Raji cells hPD-L1 or Null
  • 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.
  • Figure 13 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.
  • Figure 13A 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.
  • hPD-L1 i.e.
  • rosnilimab 158 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 remaining window for 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 .
  • the three hPD1 »hPDL1 stabilizing Nanobodies selected herein present a unique profile ( Figure 13C). 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-LI ). 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 .
  • CA19281 (SEQ ID NO: 15) was the most potent with an EC 5 o 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 14).
  • 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.
  • 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 hPD-L1 -dependent manner.
  • NFAT nuclear factor of activated T cells
  • 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.
  • 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.
  • 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.
  • rosnilimab exhibited an additive effect on hPD-L1 -induced suppression of NFAT signaling, resulting in a further decrease in luminescence.
  • 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 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.
  • 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 and removal of residues problematic for chemical and biophysical stability. However, the process should have minimal impact on target binding and biological function.
  • 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 20) and on prior knowledge of motifs potentially leading to post-translational modifications 123 , resulting in a framework-optimized sequence of A00032 (SEQ ID NO: 49).
  • CA19281 was shown to improve the K d app of PD-L1 binding to PD-1 by 2055-fold.
  • 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).
  • Single mutations toward the human sequence can then be introduced in regions outside the antigen-binding sites that are more likely to influence binding.
  • methionine 85 M85
  • threonine T
  • threonine 68 T68
  • alanine A
  • mutant libraries were generated for each problematic residue, as predicting substitutions that preserve functionality in these regions is challenging.
  • an NS motif susceptible to deamination, was found in the CDR1
  • methionine susceptible to oxidation, was present in the CDR3.
  • the N36Q and N36R mutations (for the former) and M109L, M109S, M109D, and M109R mutations (forthe latter) preserved modulator functionality in the SHP1 recruitment assay (data not shown).
  • all permitted mutations were combined into a single sequence.
  • the mutations N36Q, T68A, M85T, and M109L yielded the optimized version of A00032, designated nanobody A00235 (SEQ ID NO: 53).
  • SEQ ID NO: 53 As shown in Figure 21C, 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.
  • the current Example shows that sequence optimization of the PAM sequences disclosed herein can be performed without compromisingtheir allosteric affinity-modulating properties and pharmacological activity.
  • the sequences of various optimized nanobody variants generated in this Example encompass SEQ ID NO: 1
  • PAMs of the current invention can be formatted for half-life extension and enhanced drug-like properties, among other properties.
  • 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 (WG2012175400) 159-162 .
  • 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).
  • 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 crosslinking 165 (see Example 12).
  • 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 22E, this formatting resulted in improved potency in the SHP1 assay.
  • 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 24A-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).
  • 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 art 166 167 , among other effects.
  • the IgGI WT moiety interfered partially with the assay, as shown by the decrease in signal with high doses of IgG fused irrelevant Nb ( Figure 24C), the IgG 1 fusion of CA19281 (SEQ ID NO: 15), A00062 (SEQ ID NO: 70), strongly improved potency in the NFAT signaling assay compared to its parent.
  • 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.
  • MLR mixed lymphocyte reaction
  • PBMCs peripheral blood mononuclear cells
  • ELISA LEGEND MAXTM
  • Luminex Luminex
  • Nanobodies that stabilize the hPD-1 •hPD-LI 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.
  • DyLight647-labeled His- tagged Nanobodies hPD-1 »hPD-L1 complex-stabilizing CA18811 and CA19275, or an irrelevant Nanobody
  • a DyLight647-labeled PD-1 antibody peresolimab
  • 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 19 Epitope characterization by BLI-based epitope binning.
  • 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).
  • 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), A00115 (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 29.
  • Example 21 Epitope mapping of hPD-1 »hPD-L1 complex PAM Nbs through screening of a PD- 1 mutant library.
  • 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.
  • 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.
  • ChILL & DisCO to discover competitive, connective and allosteric Nanobodies that modulate the SOS1 »RAS protein- protein interactions and tune the nucleotide exchange rate
  • LAIR-1 leukocyte-associated Ig-like receptor-1

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Abstract

La présente divulgation concerne un modulateur contenant un domaine variable unique d'immunoglobuline (ISVD) qui se lie de manière allostérique à un épitope tridimensionnel (3D) présent sur le complexe PD-1•PDL1 (hPD-1•hPD-L1) humain. Le modulateur régule la coopérativité dans ce complexe, affectant ainsi l'affinité de liaison entre hPD-1 et hPD-L1 et/ou des voies de signalisation en aval. Plus particulièrement, le modulateur allostérique selon la présente invention induit sélectivement une coopérativité positive dans le complexe hPD-1•hPD-L1, ce qui permet de supprimer les réponses immunitaires de manière à limitation spatiotemporelle. En conséquence, de tels modulateurs allostériques sont utiles en tant qu'agents thérapeutiques pour des maladies inflammatoires, telles que des maladies auto-immunes, des maladies allergiques ou une maladie du greffon contre l'hôte (GVHD).
PCT/EP2025/068744 2024-07-01 2025-07-01 Liants du complexe pd-1•pd-l1 et leur utilisation Pending WO2026008665A1 (fr)

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