HK1207387B - Anti-pd-l1 antibodies and their use to enhance t-cell function - Google Patents

Description

Anti-PD-L1 antibodies and their use for enhancing T cell function

The present application is a divisional application of Chinese patent application No. 200980149532.9, filed on December 8, 2009, and entitled “Anti-PD-L1 antibodies and their use for enhancing T cell function”.

Related applications

This application claims the benefit of priority under 35 USC 119(e) to U.S. Provisional Application No. 61/121,092, filed December 9, 2008, the disclosure of which is incorporated herein by reference in its entirety.

Field of the Invention

The present invention relates generally to immune function and to enhancing T cell function, including upregulating cell-mediated immune responses, and to the treatment of conditions in which T cell dysfunction occurs.

Background of the Invention

Costimulation, or providing two different signals to T cells, is a widely accepted model of lymphocyte activation of resting T lymphocytes by antigen presenting cells (APCs). Lafferty et al., Aust. J. Exp. Biol. Med. Sci. (Australian Journal of Experimental Biology and Medical Science) 53 : 27-42 (1975). This model further provides for the discrimination of self from non-self and immune tolerance. Bretscher et al., Science (Science) 169 : 1042-1049 (1970); Bretscher, PA, PNAS USA 96 : 185-190 (1999); Jenkins et al., J. Exp. Med. (Journal of Experimental Medicine) 165 : 302-319 (1987). After recognition of heterologous antigenic peptides presented in the context of the major histocompatibility complex (MHC), the primary signal or antigen-specific signal is transduced by the T cell receptor (TCR). Secondary or costimulatory signals are delivered to T cells via costimulatory molecules expressed on antigen-presenting cells (APCs), inducing T cells to promote clonal expansion, cytokine secretion, and effector function. Lenschow et al., Ann. Rev. Immunol. 14 :233 (1996). Without costimulation, T cells do not sense antigenic stimulation and do not initiate an effective immune response. This can also lead to exhaustion or tolerance to heterologous antigens.

The simple two-signal model may be an oversimplification because the intensity of the TCR signal actually has a quantitative effect on T cell activation and differentiation. Viola et al., Science 273 : 104-106 (1996); Sloan-Lancaster, Nature 363 : 156-159 (1993). In addition, if the TCR signal intensity is high enough, T cell activation can occur even in the absence of costimulatory signals. More importantly, T cells receive both positive and negative secondary costimulatory signals. The regulation of such positive and negative signals is important for maximizing the protective immune response to the host while maintaining immune tolerance and preventing autoimmunity. Negative secondary signals seem to be necessary for inducing T cell tolerance, while positive signals promote the activation of T cells. Although the simple two-signal model still provides an effective explanation for immature lymphocytes, the host's immune response is a dynamic process, and costimulatory signals can also be provided to T cells exposed to antigens.

The mechanism of co-stimulation has therapeutic significance because it has been shown that the manipulation of co-stimulatory signals provides a means of enhancing or terminating cell-based immune responses. Recently, it has been found that T cell dysfunction or anergy occurs simultaneously with the induction and sustained expression of the inhibitory receptor programmed death 1 polypeptide (PD-1). Therefore, therapeutic targeting of PD-1 and other molecules that transmit signals by interacting with PD-1, such as programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2), is an area of strong interest. Inhibition of PD-L1 signaling has been proposed as a means of enhancing T cell immunity for the treatment of cancer (e.g., tumor immunity) and infection (including acute and chronic (e.g., persistent) infection). However, because there is still a need to commercialize the best treatment for targets of this pathway, there is a significant unmet medical need.

SUMMARY OF THE INVENTION

The present application provides anti-PD-L1 antibodies, including nucleic acids encoding such antibodies and compositions containing such antibodies, as well as their use for enhancing T cell function to upregulate cell-mediated immune responses and providing treatments for T cell dysfunction disorders, including infections (e.g., acute and chronic) and tumor immunity.

In one embodiment, the invention provides an isolated heavy chain variable region polypeptide comprising HVR-H1, HVR-H2, and HVR-H3 sequences, wherein:

(a) the HVR-H1 sequence is GFTFSX ₁ SWIH (SEQ ID NO: 1);

(b) the HVR-H2 sequence is AWIX ₂ PYGGSX ₃ YYADSVKG (SEQ ID NO: 2);

(c) the HVR-H3 sequence is RHWPGGFDY (SEQ ID NO: 3);

Furthermore, _X1 is D or G; _X2 is S or L; _X3 is T or S.

In a specific aspect, _X1 is D; _X2 is S and _X3 is T. In another aspect, the polypeptide further comprises a variable region heavy chain framework sequence juxtaposed between the HVRs according to the formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4). In yet another aspect, the framework sequence is derived from a human consensus framework sequence. In yet another aspect, the framework sequence is a VH subgroup III consensus framework. In yet another aspect, at least one of the framework sequences is as follows:

HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:4)

HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO: 5)

HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:6)

HC-FR4 is WGQGTLVTVSA (SEQ ID NO: 7).

In yet another aspect, the heavy chain polypeptide is further combined with a variable region light chain comprising HVR-L1, HVR-L2, and HVR-L3, wherein:

(a) the HVR-L1 sequence is RASQX ₄ X ₅ X ₆ TX ₇ X ₈ A (SEQ ID NOs: 8);

(b) the HVR-L2 sequence is SASX ₉ LX ₁₀ S (SEQ ID NOs: 9);

(c) the HVR-L3 sequence is QQX ₁₁ X ₁₂ X ₁₃ X ₁₄ PX ₁₅ T (SEQ ID NOs: 10);

wherein: _X4 is D or V; _X5 is V or I; _X6 is S or N; _X7 is A or F; _X8 is V or L; _X9 is F or T; _X10 is Y or A; _X11 is Y, G, F, or S; _X12 is L, Y, F, or W; _X13 is Y, N, A, T, G, F, or I; _X14 is H, V, P, T, or I; and _X15 is A, W, R, P, or T.

In yet another aspect, _X4 is D; _X5 is V; _X6 is S; _X7 is A; X8 is V; _X9 is F; _X10 is Y; _X11 is Y; _X12 is L; _X13 is Y; _X14 is H; _X15 is A. In yet another aspect, the light chain further comprises a variable region light chain framework sequence juxtaposed between the HVRs according to the formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR- _L2 )-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequence is derived from a human consensus framework sequence. In yet another aspect, the framework sequence is a VLκI consensus framework. In yet another aspect, at least one of the framework sequences is as follows:

LC-FR1 is DIQMTQSPSSLSSASVGDRVTITC (SEQ ID NO: 11);

LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO: 12);

LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 13);

LC-FR4 is FGQGTKVEIKR (SEQ ID NO: 14).

In another embodiment, the present invention provides an isolated anti-PD-L1 antibody or antigen-binding fragment comprising heavy and light chain variable region sequences, wherein:

(a) the heavy chain comprises HVR-H1, HVR-H2 and HVR-H3, and wherein:

(i) the HVR-H1 sequence is GFTFSX ₁ SWIH (SEQ ID NO: 1);

(ii) the HVR-H2 sequence is AWIX ₂ PYGGSX ₃ YYADSVKG (SEQ ID NO: 2);

(iii) the HVR-H3 sequence is RHWPGGFDY (SEQ ID NO: 3); and

(b) the light chain comprises HVR-L1, HVR-L2, and HVR-L3, and wherein:

(i) the HVR-L1 sequence is RASQX ₄ X ₅ X ₆ TX ₇ X ₈ A (SEQ ID NOs: 8);

(ii) the HVR-L2 sequence is SASX ₉ LX ₁₀ S (SEQ ID NOs: 9); and

(iii) the HVR-L3 sequence is QQX ₁₁ X ₁₂ X ₁₃ X ₁₄ PX ₁₅ T (SEQ ID NOs: 10);

wherein: _X1 is D or G; _X2 is S or L; _X3 is T or S; _X4 is D or V; _X5 is V or I; _X6 is S or N; _X7 is A or F; _X8 is V or L; _X9 is F or T; _{X10 is Y or A; X11 is} Y, G, F, or S; _X12 is L, Y, F, or W; _X13 is Y, N, A, T, G, F, or I; _X14 is H, V, P, T, or I; and _X15 is A, W, R, P, or T.

In one specific aspect, _X1 is D; _X2 is S and _X3 is T. In another aspect, _X4 is D; _X5 is V; _X6 is S; _X7 is A; X8 is V; _X9 is F; _X10 is Y; _X11 is Y; _X12 is L; _X13 is Y; _X14 is H _{; X15} is A. In yet another aspect, _X1 is D; _X2 is S and _X3 is T, _X4 is D; _X5 is V; _X6 is S; _X7 is A; _X8 is V; _X9 is F; _X10 is Y; _X11 is Y; _X12 is L; _X13 is Y; _X14 is H and _X15 is A.

In another aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In another aspect, the framework sequences are derived from human consensus framework sequences. In another aspect, the heavy chain framework sequences are derived from Kabat subgroup I, II, or III sequences. In another aspect, the heavy chain framework sequences are VH subgroup III consensus frameworks. In another aspect, one or more of the heavy chain framework sequences are as follows:

In another aspect, the light chain framework sequence is derived from a Kabat κI, II, III, or IV subtype sequence. In another aspect, the light chain framework sequence is a VLκI consensus framework. In another aspect, one or more of the light chain framework sequences are as follows:

In yet another specific aspect, the antibody further comprises a human or mouse constant region. In yet another aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In yet another specific aspect, the human constant region is IgG1. In yet another aspect, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In yet another aspect, the mouse constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, the minimal effector function is produced by an "effector-less Fc mutation" or aglycosylation. In yet another embodiment, the Fc mutation of the worse effector is an N297A or D265A/N297A substitution in the constant region.

In yet another embodiment, the present invention provides an anti-PD-L1 antibody comprising heavy and light chain variable region sequences, wherein:

(a) the heavy chain further comprises HVR-H1, HVR-H2 and HVR-H3 sequences that have at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO: 15), AWISPYGGSTYYADSVKG (SEQ ID NO: 16) and RHWPGGFDY (SEQ ID NO: 3), respectively, or

(b) the light chain further comprises HVR-L1, HVR-L2 and HVR-L3 sequences that have at least 85% sequence identity to RASQDVSTAVA (SEQ ID NO: 17), SASFLYS (SEQ ID NO: 18) and QQYLYHPAT (SEQ ID NO: 19), respectively.

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In another aspect, the framework sequences are derived from human consensus framework sequences. In another aspect, the heavy chain framework sequence is derived from a Kabat subgroup I, II, or III sequence. In another aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In another aspect, one or more of the heavy chain framework sequences are as follows:

In another aspect, the light chain framework sequence is derived from a Kabat κI, II, III, or IV subtype sequence. In another aspect, the light chain framework sequence is a VLκI consensus framework. In another aspect, one or more of the light chain framework sequences are as follows:

In yet another specific aspect, the antibody further comprises a human or murine constant region. In yet another aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In yet another specific aspect, the human constant region is IgG1. In yet another aspect, the murine constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In yet another aspect, the murine constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, the minimal effector function is produced by a "poor effector Fc mutation" or deglycosylation. In yet another embodiment, the poor effector Fc mutation is an N297A or D265A/N297A substitution in the constant region.

In yet another embodiment, the present invention provides an isolated anti-PD-L1 antibody comprising heavy and light chain variable region sequences, wherein:

(a) the heavy chain sequence has at least 85% sequence identity with the following heavy chain sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO: 20), or

(b) the light chain sequence has at least 85% sequence identity with the following light chain sequence:

DIQMTQSPSSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 21).

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In another aspect, the framework sequences are derived from human consensus framework sequences. In another aspect, the heavy chain framework sequences are derived from Kabat subgroup I, II or III sequences. In another aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In another aspect, one or more of the heavy chain framework sequences are as follows:

In another aspect, the light chain framework sequence is derived from a Kabat kappa I, II, III or IV subtype sequence. In another aspect, the light chain framework sequence is a VL kappa I consensus framework. In another aspect, one or more of the light chain framework sequences are as follows:

In yet another specific aspect, the antibody further comprises a human or murine constant region. In yet another aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In yet another specific aspect, the human constant region is IgG1. In yet another aspect, the murine constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In yet another aspect, the murine constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, the minimal effector function results from production in prokaryotic cells. In yet another specific aspect, the minimal effector function results from a "poorer effector Fc mutation" or deglycosylation. In yet another embodiment, the poorer effector Fc mutation is an N297A or D265A/N297A substitution in the constant region.

In yet another embodiment, the present invention provides a composition comprising any one of the anti-PD-L1 antibodies described above in combination with at least one pharmaceutically acceptable carrier.

In yet another embodiment, the present invention provides an isolated nucleic acid encoding a light chain or heavy chain variable region sequence of an anti-PD-L1 antibody, wherein:

(a) the heavy chain further comprises HVR-H1, HVR-H2, and HVR-H3 sequences that have at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO: 15), AWISPYGGSTYYADSVKG (SEQ ID NO: 16), and RHWPGGFDY (SEQ ID NO: 3), respectively, and

(b) the light chain further comprises HVR-L1, HVR-L2 and HVR-L3 sequences that have at least 85% sequence identity to RASQDVSTAVA (SEQ ID NO: 17), SASFLYS (SEQ ID NO: 18) and QQYLYHPAT (SEQ ID NO: 19), respectively.

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In one aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In another aspect, the framework sequences are derived from human consensus framework sequences. In another aspect, the heavy chain framework sequence is derived from a Kabat subgroup I, II, or III sequence. In another aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In another aspect, one or more of the heavy chain framework sequences are as follows:

In another aspect, the light chain framework sequence is derived from a Kabat κI, II, III, or IV subtype sequence. In another aspect, the light chain framework sequence is a VLκI consensus framework. In another aspect, one or more of the light chain framework sequences are as follows:

In yet another specific aspect, the antibody further comprises a human or murine constant region. In yet another aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In yet another specific aspect, the human constant region is IgG1. In yet another aspect, the murine constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In yet another aspect, the murine constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, the minimal effector function results from production in prokaryotic cells. In yet another specific aspect, the minimal effector function results from a "poorer effector Fc mutation" or deglycosylation. In yet another embodiment, the poorer effector Fc mutation is an N297A or D265A/N297A substitution in the constant region.

In another aspect, the nucleic acid further comprises a vector suitable for expressing the nucleic acid encoding any of the aforementioned anti-PD-L1 antibodies. In another specific aspect, the vector further comprises a host cell suitable for expressing the nucleic acid. In another specific aspect, the host cell is a eukaryotic cell or a prokaryotic cell. In another specific aspect, the eukaryotic cell is a mammalian cell, such as a Chinese hamster ovary (CHO).

In yet another embodiment, the present invention provides a method for preparing an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising culturing a host cell containing a nucleic acid in a form suitable for expression, the nucleic acid encoding any of the aforementioned anti-PD-L1 antibodies or antigen-binding fragments, under conditions suitable for production of such antibodies or fragments, and recovering the antibody or fragment.

In yet another embodiment, the present invention provides a composition comprising an anti-PD-L1 antibody or antigen-binding fragment thereof provided herein and at least one pharmaceutically acceptable carrier.

In yet another embodiment, the invention provides an article of manufacture comprising a container containing a therapeutically effective amount of a composition described herein and a package insert directing use for treating a T-cell dysfunction disorder.

In another embodiment, the present invention provides an article of manufacture comprising a combination of any of the above-described anti-PD-L1 compositions and at least one BNCA molecule. In one aspect, the BNCA molecule is an antibody, an antigen-binding antibody fragment, a BNCA oligopeptide, a BNCA RNAi, or a BNCA small molecule. In another aspect, the B7 negative costimulatory molecule is selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, B7.1, B7-H3, and B7-H4.

In yet another embodiment, the article of manufacture comprises a combination of any of the anti-PD-L1 compositions described above and a chemotherapeutic agent. In one aspect, the chemotherapeutic agent is gemcitabine.

In another embodiment, the present invention provides a product comprising a combination of any of the anti-PD-L1 antibodies described above and an agonist of one or more positive co-stimulatory molecules. In one aspect, the positive co-stimulatory molecule is a B7 family co-stimulatory molecule. In another aspect, the positive co-stimulatory molecule is selected from the group consisting of the following: CD28, CD80, CD86, ICOS/ICOSL. In another aspect, the positive co-stimulatory molecule is a TNFR family co-stimulatory molecule. In another aspect, the TNFR co-stimulatory molecule is selected from the group consisting of the following: OX40/OX40L, 4-1BB/4-1BBL, CD27/CD27L, CD30/CD30L and HVEM/LIGHT, and soluble fragments thereof, constructs and agonistic antibodies.

In yet another embodiment, the present invention provides an article of manufacture comprising any of the above-described anti-PD-L1 antibodies in combination with one or more antibiotics. In one aspect, the antibiotic is selected from the group consisting of an antiviral agent, an antibacterial agent, an antifungal agent, and an antiprotozoal agent.

In another aspect, the antiviral agent is selected from the group consisting of a reverse transcriptase inhibitor, a protease inhibitor, an integrase inhibitor, an entry or fusion inhibitor, a maturation inhibitor, a viral release inhibitor, an immune response enhancer, an antiviral synergist, a vaccine, a hepatic agonist, and an herbal remedy. In yet another aspect, the combination comprises one or more types of antiviral agents.

In yet another embodiment, the invention provides an article of manufacture comprising any one of the anti-PD-L1 antibodies described above in combination with one or more vaccines.

In another embodiment, the present invention provides a method for enhancing T cell function, comprising administering an effective amount of any one of the anti-PD-L1 antibodies or compositions described above. In one aspect, the anti-PD-L1 antibody or composition renders dysfunctional T cells non-dysfunctional.

In another embodiment, the present invention provides a method for treating a T cell dysfunction disorder comprising administering a therapeutically effective amount of any one of the anti-PD-L1 antibodies or compositions described above. In a specific aspect, the T cell dysfunction disorder is an infection or tumor immunity. In another aspect, the infection is acute or chronic. In another aspect, the chronic infection is a persistent, latent infection or a slow infection. In another aspect, the chronic infection is caused by a pathogen selected from the group consisting of bacteria, viruses, fungi and protozoa. In another aspect, the level of the pathogen in the host is reduced. In another aspect, the method further comprises treatment with a vaccine. In another aspect, the method further comprises treatment with an antibiotic. In another aspect, the pathogen is a bacterium and the method further comprises administering an antibacterial agent. In another aspect, the bacterium is selected from the group consisting of Mycobacterium spp., Salmonella spp., Listeria spp., Streptococcus spp., Haemophilus spp., Neisseria spp., Klebsiella spp., Borrelia spp., Bacterioides fragillis, Treponema spp., and Helicobacter pylori. In another aspect, the pathogen is a virus, and the method further comprises administering an antiviral agent. In another aspect, the virus is selected from the group consisting of hepatitis B virus, hepatitis C virus (hepatitis-B, -C), herpes simplex virus-I, -II, human immunodeficiency virus-I, -II, cytomegalovirus, Epstein Barr virus, human papillomavirus, human T lymphotrophic viruses, -I, -II, varicalla zoster virus. In another aspect, the pathogen is a fungus, and the method further comprises administering an antifungal agent. In another aspect, the condition is selected from the group consisting of aspergillosis, blastomycosis, candidiasis albicans, coccidioidomycosis immitis, histoplasmosis, paracoccidiomycosis, and microsporidiosis. In another aspect, the pathogen is a protozoan and the method further comprises administering an antiprotozoal agent. In yet another aspect, the disorder is selected from the group consisting of leishmaniasis, plasmodiosis (i.e., malaria), cryptosporidiosis, toxoplasmosis, trypanosomiasis, and helminth infection, including those caused by trematodes (e.g., schistosomiasis), cestodes (e.g., echinococcosis), and nemotodes (e.g., trchinosis, ascariasis, filariosis, and strongylodiosis).

In another aspect, the T cell dysfunction disorder is tumor immunity. In another aspect, the PD-L1 antibody or composition and the treatment regimen combination further include conventional therapy, the conventional therapy is selected from the group consisting of the following: radiotherapy, chemotherapy, targeted therapy, immunotherapy, hormone therapy, angiogenesis inhibition and palliative care (palliativecare). In another specific aspect, the chemotherapy treatment is selected from the group consisting of the following: gemcitabine (gemcitabine), cyclophosphamide (cyclophosphamide), doxorubicin (doxorubicin), paclitaxel (paclitaxel), cisplatin (cisplatin). In yet another specific aspect, the tumor immunity is caused by a cancer selected from the group consisting of breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma, bladder cancer, kidney cancer, liver cancer, salivary cancer, stomach cancer, gliomas, thyroid cancer, thymic cancer, epithelial cancer, head and neck cancers, gastric and pancreatic cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic illustration depicting T cell co-stimulation by B7 family cell surface molecules.

FIG2 is a schematic diagram showing the experimental design of the PMEL/B16 T cell stimulation assay.

Figure 3 is a bar graph showing the effect of anti-PD-L1 Ab on antigen-specific T cell function by enhancing IFN-γ production in PMEL CD8+ T cells in response to the melanocyte peptide gp100. Both the percentage of CD8+ T cells producing IFN-γ and their IFN-γ production levels increased during stimulation in the presence of anti-PD-L1 antibodies.

FIG4 is a bar graph showing the effect of anti-PD-L1 Ab on antigen-specific T cell function by enhancing the proliferation of Ova-specific CD4+ T cells, which was achieved by anti-PD-L1 Ab YW243.55.S1 after secondary stimulation with Ova-pulsed A20 B cells/mPD-L1 APCs.

Figure 5 is a series of FACS images showing that the proliferation of human CD8 T cells is enhanced in a mixed lymphocyte reaction by the anti-PD-L1 antibody YW243.55S1. The percentage of proliferating cells measured by CFSE intensity by dilution is also reported.

Figure 6 is a schematic diagram of the experimental design for the treatment of chronic LCMV with the chimeric anti-PD-L1 Ab YW243.55S70. Arrows indicate the timing of the six doses of anti-PD-L1 starting 14 days after infection with 2 x ¹⁰⁶ pfu clone 13 LCMV.

Figures 7A and 7B are graphs showing enhanced CD8 effector function in ex vivo cells following treatment of chronic LCMV infection with anti-PD-L1 Ab YW243.55.S70. Blocking PD-L1 by YW243.55.S70 increased degranulation of CD8 ⁺ T cells (as measured by an increase in surface CD107A) (Figure 7A) and increased the production of IFN-γ cells in response to the LCMV peptide gp33 (Figure 7B). The frequency of gp33-specific cells was shown by staining with H2-Db gp33 pentamer.

Figures 8A and 8B show the reduction of blood and tissue LCMV titers in chronic LCMV infection after in vivo treatment with anti-PD-L1 antibodies. In Figure 8A, viral titers from the various tissues indicated were analyzed on days 21 and 28 (one week and two weeks after Ab treatment, respectively). In Figure 8B, serum viral titers were analyzed on days 0, 7, 14, 21, and 28, with LCMV inoculation occurring on day 0 and treatment starting on day 14.

Figure 9A shows a significant reduction in MC38.Ova colon cancer tumor growth due to the application of anti-PD-L1 antibodies after therapeutic treatment of established tumors (treatment started on day 14 when the tumors were 250 mm ³ ). Figure 9B is a histogram showing the surface level of PD-L1 expression on MC38.Ova cells in tissue culture measured by flow cytometry. PD-L2 is not expressed by MC38.Ova cells.

FIG10 is a graph showing the effects of PD-L1 blockade therapy alone and in combination with anti-VEGF or gemcitabine on MC38.Ova tumor growth in C57BL/6 mice.

Figure 11A-B are the heavy chain and light chain variable region sequences of 11 anti-PD-L1 antibodies identified by phage display, respectively. The shaded bars indicate differently defined CDRs, while the boxed areas indicate the extent of the HVRs.

Detailed Description of the Preferred Embodiments

All references mentioned herein are expressly incorporated by reference.

General Technology

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. These techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Animal Cell Culture (R.I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987, and regularly updated); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); A Practical Guide to Molecular Cloning (Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001).

I. Host Immunity

A. Lymphocyte development and activation

The two main types of lymphocytes in humans are T (thymus-derived) and B (bone marrow-derived). These cells originate from hematopoietic stem cells in the bone marrow and fetal liver that have been committed to the lymphoid development pathway. The descendants of these stem cells mature along different pathways into B or T lymphocytes. In humans, B lymphocyte development occurs entirely within the bone marrow. T cells, on the other hand, develop from immature precursors that leave the bone marrow and travel through the bloodstream to the thymus, where they proliferate and differentiate into mature T lymphocytes.

Mature lymphocytes that emerge from the thymus or bone marrow are in a resting or "quiescent" state, that is, they are mitotically inactive. Upon dispersal into the bloodstream, these "immature" or "virgin" lymphocytes migrate to a variety of secondary or peripheral lymphoid organs, such as the spleen, lymph nodes, or tonsils. Most virgin lymphocytes have an inherently short lifespan and die a few days after leaving the bone marrow or thymus. However, if such cells receive a signal that an antigen is present, they can activate and undergo successive rounds of cell division. Some of the resulting progeny cells then revert to a resting state and become memory lymphocytes—B and T cells—which are essentially sensitized upon further encounter with the inciting allergen. Other descendants of activated virgin lymphocytes are effector cells, which survive only a few days but carry out specific defensive activities.

Lymphocyte activation refers to an ordered series of events by which resting lymphocytes, when stimulated, undergo division and produce offspring, some of which become effector cells. The complete response includes induction of cell proliferation (mitogenesis) and expression of immune function. Lymphocytes are activated when specific ligands bind to receptors on their surfaces. The ligands are different for T cells and B cells, but the resulting intracellular physiological mechanisms are similar.

Some heterologous antigens themselves can induce lymphocyte activation, particularly large polymeric antigens cross-linked with surface immunoglobulins on B cells or other glycoproteins on T cells. However, most antigens are not polymeric and can not cause activation even if they are directly bound to B cells in large quantities. These more common antigens activate B cells when costimulating with nearby activated helper T-lymphocytes. This type of stimulation can come from lymphokines secreted by T cells, but its most effective transmission is by directly contacting B cells with T cell surface proteins, which interact with certain B cell surface receptors to produce secondary signals.

B. T cells

T lymphocytes do not express immunoglobulins, but instead detect the presence of foreign substances through surface proteins called T cell receptors (TCRs). These receptors recognize antigens either through direct contact or by influencing the activity of other immune cells. Along with macrophages, T cells are the main cell type involved in cell-mediated immunity.

Unlike B cells, T cells are only able to detect foreign substances in specific environments. Specifically, T lymphocytes will recognize foreign proteins only when they are first cleaved into small peptides, which are then displayed on the surface of a second host cell, called an antigen presenting cell (APC). Many types of host cells are capable of presenting antigens under certain conditions, but certain types are more specifically suited for this purpose and are particularly important in controlling T cell activity, including macrophages and other B cells. Antigen presentation depends in part on specific proteins, called major histocompatibility complex (MHC) proteins, which are present on the surface of presenting cells. Therefore, in order to stimulate cell-mediated immunity, foreign peptides must be presented to T cells in combination with MHC peptides, and this combination must be recognized by the T cell receptor.

There are two main T cell subtypes, cytotoxic T lymphocytes ( _Tc cells or CTLs) and helper T cells ( _TH ) cells, which can be roughly identified based on the cell surface expression markers CD8 and CD4. _Tc cells are important in viral defense and can directly kill viruses by recognizing viral peptides expressed on the surface of certain cells. _TH cells promote the proliferation, maturation and immune function of other cell types, such as lymphokine secretion, which controls the activity of B cells, macrophages and cytotoxic T cells. Virgin and memory T lymphocytes are usually in a resting state, in which they do not show significant helper or cytotoxic activity. When activated, these cells undergo several rounds of mitosis to produce daughter cells. Some of these daughter cells return to a resting state as memory cells, but others become effector cells, actively expressing helper or cytotoxic activity. These daughter cells are similar to their parents: CD4+ cells can only produce CD4+ offspring, while CD8+ cells can only produce CD8+ offspring. Effector T cells express cell surface markers that are not expressed on resting T cells, such as CD25, CD28, CD29, CD40L, transferrin receptor, and class II MHC proteins. When the activating stimulus is withdrawn, the cytotoxic or helper activity gradually subsides over a period of days as the effector cells die or return to a quiescent state.

Similar to the activation of B cells, T lymphocytes also require two types of simultaneous stimulation for the response to most antigens. The first is an antigen that, if properly displayed on antigen-presenting cells by MHC proteins, can be recognized and bound by the T cell receptor. Although this antigen-MHC complex does send signals into the cell, it is usually not enough to cause T cell activation. Complete activation, such as occurs with helper T cells, requires costimulation together with other specific ligands, which are called co-stimulatory factors and are expressed on the surface of antigen-presenting cells. On the other hand, the activation of cytotoxic T cells usually requires IL-2, a cytokine secreted by activated helper T cells.

C. Immune response

The three main functional properties that distinguish the mammalian immune system from other body defenses include: (1) specificity - the ability to uniquely recognize and respond or not respond among a large number of target molecules, (2) recognition - the ability to distinguish self from non-self, thereby coexisting peacefully with a myriad of proteins and other organic molecules, yet still responding vigorously to foreign substances introduced into the body, and (3) memory - the ability to model through experience so that subsequent encounters with specific foreign pathogens will trigger a more rapid and robust response than the initial encounter. When one or more of these functions is disrupted, physiological disorders result.

Virgin lymphocytes are continuously released into the periphery from primary lymphoid organs, each carrying surface receptors capable of antigen binding. Antigen binding in B cells is mediated by surface-bound immunoglobulins, while in T cells it is mediated by T cell receptors. However, when virgin lymphocytes are activated, they proliferate to produce daughter cells, which can then undergo further cycles of activation and proliferation. The speed and intensity of the response to a given antigen are largely determined by clonal selection: the larger the daughter cells or clones specific for a particular antigen, the greater the number of cells capable of recognizing and participating in the immune response. Each immune response is complex and intricately regulated by a sequence of events involving several cell types. When an immunogen enters the body and encounters a specialized type of cell, called an antigen-presenting cell (APC), it is triggered. These APCs capture minute amounts of the immunogen and present it in a form that can be recognized by antigen-specific helper T lymphocytes. The helper T cells are then activated and, in turn, promote the activation of other types of lymphocytes, such as B cells or cytotoxic T cells. The activated lymphocytes then proliferate and perform their specific effector functions. At each stage of this process, lymphocytes and APCs communicate with each other through direct contact or through the secretion of regulatory cytokines.

Foreign antigens captured by APCs undergo a series of changes called antigen processing. Such processing (particularly of protein immunogens) involves denaturation and partial proteolytic digestion, whereby the immunogen is cut into short peptides. The limited number of peptides produced are then non-covalently bound to class II MHC proteins and transported to the surface of the APCs, a process known as antigen presentation. CD4+ helper T lymphocytes in direct contact with APCs can be activated, but they are only activated when they express T cell receptor proteins that can recognize and bind to the specific peptide-MHC complex presented by the APCs.

Helper T ( _TH ) cells are the main orchestrators of the immune response because they are needed to activate two other lymphoid effector cells: cytotoxic T (Tc) cells and antibody-secreting plasma cells. _TH activation occurs early in the immune response and requires at least two signals. One signal is provided by the binding of the T cell antigen receptor to the antigenic peptide-MHC complex transmitted by the CD3 protein complex on the surface of the APC, while the second co-stimulatory signal through the APC is believed to come from the binding of an independent signaling protein on the surface of the T cell to a specific ligand on the APC. One known such interaction is the T cell protein CD28 and a family of APC surface proteins known as B7. Other surface protein pairs can also mediate co-stimulation. The process of co-stimulation is described in more detail later. It is believed that the anti-PD-L1 antibodies of the present invention enhance co-stimulation by antagonizing the negative co-stimulatory signal provided by signal transduction through PD-L1.

In short, two signals induce helper T cells to begin secreting the cytokine interleukin-2 (IL-2) and also begin to express specific high-affinity IL-2 receptors on their surface. IL-2 is a highly powerful mitogenic factor for T lymphocytes and is essential to the proliferative response of activated T cells. The effect of IL-2 on the cells that secrete it is a phenomenon known as the autocrine effect. It has also been shown that even if T cells have received two signals, they will not proliferate if their own IL-2 receptors are blocked. IL-2 can also act on the cells in the immediate vicinity through the so-called paracrine effect. This effect is particularly important for activating Tc cells, which do not usually produce enough IL-2 to stimulate their own proliferation. In addition to IL-2, activated T _H cells secrete other cytokines and promote the growth, differentiation and function of B cells, macrophages and other cell types.

Contact between APCs and antigen-specific _TH cells also has effects on the APCs, one of the most important of which is the release of IL-1. This cytokine is believed to act in an autocrine manner to increase surface expression of class II MHC proteins and various adhesion molecules, thereby enhancing _TH cell binding and enhancing antigen presentation. Concurrently, IL-1 acts in a paracrine manner on _TH cells to promote IL-2 secretion and expression of the IL-2 receptor.

During the activation of _TH cells in the manner described above, some B cells may also bind to the immunogen through their antigen receptors, which are membrane-bound forms of antibodies that will be secreted later. Unlike T cells, B cells recognize the immunogen in its free, unprocessed form. Specific antigen binding provides one type of signal that can lead to B cell activation. The second type is provided by activated _TH cells, which express proteins that help activate B cells by binding to non-immunoglobulin receptors on their surfaces. These _TH -derived signals, which act on B cells regardless of their antigen specificity, are called cofactors. These cofactors include IL-2, IL-4, and IL-6. However, help is more effectively achieved through cell-cell contact, which allows proteins on the surface of T cells to come into direct contact with those on _B cells. The most effective form of contact-mediated help occurs when a protein called CD40 ligand (CD40L), which is expressed on _TH cells only after they are activated, binds to a protein on B cells called CD40. In a process called bystander activation, contact with activated B cells can be sufficient to activate even resting B cells, even if the B cell's surface immunoglobulin has not yet bound antigen.

_Tc lymphocytes function to eliminate cells expressing foreign antigens on their surfaces (such as virally infected host cells). Most _Tc cells express CD8 rather than CD4 and therefore recognize antigens associated with class I rather than class II MHC proteins. When somatic cells are infected with viruses, some immunogenic viral proteins may be processed intracellularly, and the resulting peptides then appear on the surface as complexes with class I MHC molecules. These peptide-MHC complexes are then recognized by antigen-specific cloned T cell receptors, providing one of the two signals necessary for _Tc cell activation. This first signal alone induces the high-affinity IL-2 receptor on _Tc cells. The second signal is provided by IL-2 secreted from nearby activated T _H lymphocytes. Upon receiving both signals, the activated _Tc cell acquires cytotoxic activity, enabling it to kill the cell to which it has bound, as well as other cells bearing the same peptide-MHC class I complex. In some cases, killing occurs due to the release of specific toxins by the Tc _cell onto the target cell; in other cases, the Tc _cell induces the target cell to commit suicide through apoptosis. Activated _Tc cells also proliferate, generating additional _Tc cells with the same antigen specificity.

D. Co-stimulation through the immunoglobulin superfamily :

1. B7.1/B7.2–CD28/CTLA-4

Perhaps the best characterized T cell costimulatory pathway is signaling through B7.1 (CD80)/B7.2 (CD86)–CD28/CTLA-4 (CD152). This signaling pathway is important for T cell activation and tolerance. Karandikar et al., J. Neuroimmunol. 89 : 10-18 (1998); Oosterwegal et al., Curr. Opin. Immunol. 11 : 294-300 (1999); Salomon et al., Annu. Rev. Immunol. 19 : 225-252 (2001); Sansom, DM, Immunol. 101 : 169-177 (2000); Chambers et al., Annu. Rev. Immunol. 19 : 565-592 (2001).

B7.1 [Freeman et al., J. Exp. Med. 174 :625-631 (1991); Freedman et al., J. Immunol. 137 :3260-3267 (1987); Yokochi et al., J. Immunol. 128 :823-827 (1982)] and B7.2 [Freeman et al., Science 262 :909-911 (1993); Freeman et al., J. Exp. Med. 178 :2185-2192 (1993); Azuma et al., Nature 366 :76-79 (1993)] have dual specificity for two stimulatory receptors, CD-28 and CTLA-4. Aruffo et al., Proc. Natl. Acad. Sci. USA 84 :8573-8577 (1987); Gross et al., J. Immunol. 144 :3201-3210 (1990). CD28 is constitutively expressed on the surface of T cells [Gross et al., J. Immunol. 149 :380-388 (1992)], while CTLA-4, a higher affinity receptor, has expression rapidly upregulated upon T cell activation. Peach et al., J. Exp. Med. 180 : 2049-2058 (1994); Linsley et al., J. Exp. Med. 176 : 1595-1604 (1992); Kinsley et al., Immunity 1 : 793-801 (1994); Linsley et al., Immunity 4 : 535-543 (1996). Most APC populations express B7.2 constitutively at low levels, which is rapidly upregulated when B7.1 is induced late after activation. Freeman et al., Science 262 : 909-911 (1993); Hathcock et al., J. Exp. Med. 180 : 631-640 (1994). Previous expression and mouse knockout data for B7.2 suggest that B7.2 is a more important co-stimulatory molecule for initiating immune responses, but otherwise the two molecules have largely overlapping functions. McAdam et al., Immuno. Rev. 165 : 631-640 (1994).

CD28 interacts with B7.1 and B7.2 to transmit signals that work in concert with TCR signals to promote T cell activation. Lenschow et al., Annu. Rev. Immunol. 165 : 233-258 (1996); Lanzavecchia et al., Cell 96 : 1-4 (1999). Without TCR signals, CD28 signaling has no physiological significance. CD28 signaling regulates the threshold for T cell activation and significantly reduces the number of TCR bindings required for T cell activation. Viola et al., Science 273 : 104-106 (1996). CD28 activation maintains T cell responses by promoting T cell survival, thereby enabling cytokines to initiate T cell clonal expansion and differentiation. Thompson et al., Proc. Natl. Acad. Sci. USA 86 :1333-1337 (1989); Lucas et al., J. Immunol. 154 :5757-5768 (1995); Shahinian et al., Science 261 :609-612 (1993); Sperling et al., J. Immunol. 157 :3909-3917 (1996); Boise et al., Immunity 3 :87-98 (1995). CD28 also optimizes the response of previously activated T cells, promoting interleukin-2 (IL-2) production and T cell survival. Although some responses are independent of CD28, it is unclear whether this costimulatory independence is due to strong antigen stimulation or is dependent on other unknown costimulatory pathways.

CTLA-4 activation results in a negative signal that inhibits TCR- and CD-28-mediated signal transduction. Engagement of CTLA-4 results in inhibition of IL-2 synthesis and progression through the cell cycle and leads to termination of T cell responses. Walunas et al., Immunity 1 : 405-413 (1994); Walunas et al., J. Exp. Med. 183 : 2541-2550 (1996); Krummel et al., J. Exp. Med. 182 : 459-466 (1995); Brunner et al., J. Immunol. 162 : 5813-5820 (1999); Greenwald et al., Immunity 14 : 145-155 (2001). CTLA-4 plays an important role in regulating T cell responses, including peripheral T cell tolerance. Although it is unclear how signaling is coordinated through CTLA-4 and CD28, some possibilities include induction of immunosuppressive cytokines, external competition for CD28 binding to B7, direct antagonism of CD28 signaling, and/or TCR-mediated signaling.

Therefore, antagonizing CTLA-4 (e.g., antagonistic anti-CTLA antibodies) and/or agonizing B7.1/B7.2/CD28 can be used to enhance immune responses in the treatment of infections (e.g., acute and chronic) and tumor immunity.

2. ICOS/ICOSL signaling :

Another pathway of interaction between APCs and T cells occurs through ICOS (CD278) and ICOSL (B7-H2, CD275). ICOS/ICOSL signaling promotes T-helper cell differentiation and effector function and is particularly important for interleukin-10 (IL-10) production, but plays a more general role in regulating T cell expansion and IL-2 production, including regulatory T cells, T cell tolerance, and autoimmunity.

In contrast to CD28, ICOS is not constitutively expressed on naive T cells but is rapidly induced on T cells following TCR engagement. Hutloff et al., Nature 397 :263-266 (1999); Yoshinaga et al., Nature 402 :827-832 (1999); Beier et al., Eur. J. Immunol. 30 :3707-3717 (2000); Coyle et al., Immunity 13 :95-105 (2000); Mages et al., Eur. J. Immunol. 30 :1040-1047 (2000); McAdam et al., J. Immunol. 165 :5035-5040 (2000). This suggests that ICOS provides a co-stimulatory signal for activated T cells. Although ICOS expression is enhanced by co-stimulation with CD28 and ICOS expression decreases in the absence of B7.1 and B7.2, ICOS is not completely dependent on CD28 signaling. McAdam et al., J. Immunol. 165 : 5035-5040 (2000); Aicher et al., J. Immunol. 164 : 4689-4696 (2000); Kopf et al., J. Exp. Med. 192 : 53-61 (2000). ICOS is upregulated on T helper cell types 1 and 2 ( _TH1 and _TH2 ) during the initial stages of differentiation, but remains high on _TH2 cells and decreases on _TH1 cells. The expression pattern of ICOS on T cells in the germinal center (Beier et al., Eur. J. Immunol. 30 : 3707-3717 (2000); Mages et al., Eur. J. Immunol. 30 : 1040-1047 (2000) suggests a role for ICOS in T cell helper B cells. Functional studies have confirmed this, and even ICOS expression has been confirmed on rat B cells, although not in other species. Tezuka et al., Biochem. Biophys. Res. Commun. 276 : 335-345 (2000; McAdam et al., Nature 409 : 102-105 (2001); Dong et al., Nature 409 : 97-101 (2001); Dong et al., J. Immunol. 166 : 3659-3662 (2001); Tafuri et al., Nature 409 : 105-109 (2001).

One role of ICOS/ICOSL signaling appears to be regulation of cytokine production (e.g., IL-4, IL-13) by recently activated and effector T cells. Hutloff et al., Nature 397 :263-266 (1999); Coyle et al., Immunity 13 :95-105 (2000); Dong et al., Nature 409 :97-101 (2001). In studies of allergic airway disease, _TH2 effector function, but not _TH2 differentiation, was demonstrated by ICOS blockade. Tesciuba et al., J. Immunol. 167 :1996-2003 (2001). This suggests that ICOS may also regulate _TH1 effector function, and that _TH1 and _TH2 cytokine production can be inhibited by in vitro reactivation with ICOS-Ig fusion proteins. Kopf et al., J. Exp. Med. 192 : 53-61 (2000).

Another possible function of ICOS involves the maintenance of T _H 1 responses. In the experimental model of autoimmune encephalomyelitis (EAE) of multiple sclerosis, a myelin-specific CD4 ⁺ T cell-mediated T _H 1 disease, the results of ICOS blockade may be different when costimulation is blocked during T cell priming and then during the effector phase of EAE. Dong et al., Nature 409 : 97-101 (2001); Rottman et al., Nature Immunol. 2 : 605-611 (2001); Sporici et al., Clin. Immunol. 100 : 277-288 (2001). Myelin oligodendrocyte glycoprotein (MOG)-induced EAE is greatly aggravated in ICOS ^−/− knockout mice, with increased IFN-γ production compared to wild-type mice. Similarly, ICOS blockade during EAE induction exacerbates disease and also leads to increased IFN-γ production. Thus, ICOS blockade during sensitization leads to a polarized T _H 1 response. Interestingly, in vitro sensitization of myelin-specific TCR transgenic T cells in the presence of ICOS-Ig inhibits their ability to induce EAE, completely contrary to the results of ICOS-Ig blockade observed in vivo. Sporici et al., supra. The difference in the opposing results in vitro and in vivo is unclear, but may reflect the role of ICOS in regulating IL-10 production in T cells and effector T cells during ICOS blockade in vivo. Co-stimulation by IL-10 is very effective in enhancing IL-10 production, more effective than co-stimulation by CD28. Hutloff et al., supra. The IL-10, IL-12 regulatory loop is critical in regulating EAE, as IL-10-/-, but not IL4-/-, mice develop exacerbated EAE. Segal et al., J. Exp. Med. 187 : 537-546 (1998).

Another possible role of ICOS is to enhance T cell-dependent B cell humoral responses. ^ICOS-/- and ICOSL ^-/- mice have been shown to require ICOS for T cell-dependent B cell responses. Hutloff et al., Nature 397 :263-66 (1999); Chapoval et al., Nat. Immunol. 2 :269-74 (2001); Coyle et al., Immunity 13 :95-105 (2000); McAdam et al., Nature 409 :102-5 (2001); Tafuri et al., Nature 409 :105-9 (2001); Suh et al., Nat. Immunol. 4 :899-906 (2003). ICOS ^−/− mice also display reduced germinal center formation in response to primary immunization, severe defects in germinal center formation in response to secondary stimulation, and defects in IgG class switching. The function of ICOS in T:B cell interactions was further validated by the identification of homozygous deletion of ICOS in T cells from patients with adult-onset common variable immunodeficiency disease. Grimbacher et al., Nat. Immunol. 4 : 261-68 (2003).

Therefore, agonistic ICOS/ICOSL (eg, agonistic anti-ICOS antibodies, soluble ICOS/ICOSL ligands) can be used to enhance immune responses in the treatment of infection (eg, acute and chronic) and/or tumor immunity.

3. PD-1 pathway :

Important negative co-stimulatory signals regulating T cell activation are provided by the programmed death-1 receptor (PD-1) (CD279) and its ligand-binding partners, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273). The negative regulatory role of PD-1 was revealed by the autoimmunity-prone PD-1 knockout (Pdcd1 ^−/− ). Nishimura et al., Immunity 11 :141-51 (1999); Nishimura et al., Science 291 :319-22 (2001). PD-1 associates with CD28 and CTLA-4 but lacks the membrane-adjacent cysteines that allow homodimerization. The cytoplasmic domain of PD-1 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM, V/IxYxxL/V). PD-1 binds only to PD-L1 and PD-L2. Freeman et al., J. Exp. Med. 192 : 1-9 (2000); Dong et al., Nature Med. 5 : 1365-1369 (1999); Latchman et al., Nature Immunol. 2 : 261-268 (2001); Tseng et al., J. Exp. Med. 193 : 839-846 (2001).

PD-1 can be expressed on T cells, B cells, natural killer T cells, activated monocytes and dendritic cells (DCs). PD-1 is expressed by activated human CD4 ⁺ and CD8 ⁺ T cells, B cells and myeloid cells, but not by unstimulated these cells. This contrasts with the more restricted expression of CD28 and CTLA-4. Nishimura et al., Int. Immunol. 8 : 773-80 (1996); Boettler et al., J. Virol. 80 : 3532-40 (2006). At least four variants of PD-1 have been cloned from activated human T cells, including transcripts lacking (i) exon 2, (ii) exon 3, (iii) exons 2 and 3 or (iv) exons 2 to 4. Nielsen et al., Cell. Immunol. 235 : 109-16 (2005). With the exception of PD-1Δex3, all variants are expressed at similar levels as full-length PD-1 in resting peripheral blood mononuclear cells (PBMCs). When human T cells are activated with anti-CD3 and anti-CD28, the expression of all variants is significantly induced. The PD-1Δex3 variant lacks a transmembrane domain and is similar to soluble CTLA-4, which plays an important role in autoimmunity. Ueda et al., Nature 423 : 506-11 (2003). This variant is enriched in the synovial fluid and serum of patients with rheumatoid arthritis. Wan et al., J. Immunol. 177 : 8844-50 (2006).

The difference between the two PD-1 ligands lies in their expression patterns. PD-L1 is constitutively expressed on mouse T and B cells, CDs, macrophages, mesenchymal stem cells, and bone marrow-derived mast cells. Yamazaki et al., J. Immunol. 169 : 5538-45 (2002). PD-L1 is expressed on a wide range of non-hematopoietic cells (e.g., cornea, lung, vascular epithelium, liver non-parenchymal cells, mesenchymal stem cells, pancreatic islets, placental syncytiotrophoblasts, keratinocytes) [Keir et al., Annu. Rev. Immunol. 26: 677-704 (2008)] and is upregulated upon activation of many cell types. Type I and type II interferons (IFNs) upregulate PD-L1. Eppihimer et al., Microcirculation 9 :133-45 (2002); Schreiner et al., J. Neuroimmunol. 155 :172-82 (2004). When MyD88, TRAF6, and MEK are inhibited, PD-L1 expression in cells is reduced. Liu et al., Blood 110 :296-304 (2007). JAK2 is also involved in PD-L1 induction. Lee et al., FEBS Lett. 580 :755-62 (2006); Liu et al., Blood 110 :296-304 (2007). Loss or inhibition of phosphatase and tensin homolog (PTEN), a cellular phosphatase that modifies phosphatidylinositol 3-kinase (PI3K) and Akt signaling, increases post-transcriptional PD-L1 expression in cancer. Parsa et al., Nat. Med. 13 :84-88 (2007).

PD-L2 expression is more restricted than PD-L1. PD-L2 is induced on DCs, macrophages, and bone marrow-derived mast cells. PD-L2 is also expressed on approximately one-half to two-thirds of resting peritoneal B1 cells, but not on conventional B2B cells. Zhong et al., Eur. J. Immunol. (European Journal of Immunology) 37 : 2405-10 (2007). PD-L2+B1 cells bind phosphatidylcholine and may be important for innate immune responses against bacterial antigens. The induction of PD-L2 by IFN-γ is partially dependent on NF-κB. Liang et al., Eur. J. Immunol. (European Journal of Immunology) 33 : 2706-16 (2003). PD-L2 can also be induced on monocytes and macrophages by GM-CF, IL-4, and IFN-γ. Yamazaki et al., J. Immunol. 169 :5538-45 (2002); Loke et al., PNAS 100 :5336-41 (2003).

PD-1 signaling typically has a greater effect on cytokine production than on cell proliferation, and has a significant effect on the production of IFN-γ, TNF-α, and IL-2. PD-1-mediated inhibitory signaling also depends on the intensity of TCR signaling, delivering greater inhibition at low levels of TCR stimulation. This reduction can be overcome by costimulation via CD28 [Freeman et al., J. Exp. Med. (Journal of Experimental Medicine) 192 : 1027-34 (2000)] or the presence of IL-2 [Carter et al., Eur. J. Immunol. (European Journal of Immunology) 32 : 634-43 (2002)].

There is increasing evidence that signaling through PD-L1 and PD-L2 may be bidirectional. That is, in addition to modifying TCR or BCR signaling, signaling may also be transmitted back to cells expressing PD-L1 and PD-L2. Although treatment of dendritic cells with natural human anti-PD-L2 antibodies (isolated from patients with Waldenstrom's macroglobulinemia) did not find upregulation of MHC II or B7 costimulatory molecules, such cells did produce greater amounts of proinflammatory cytokines, particularly TNF-α and IL-6, and stimulate T cell proliferation. Nguyen et al., J. Exp. Med. (Journal of Experimental Medicine) 196 : 1393-98 (2002). Treatment of mice with this antibody also (1) enhanced resistance to transplated b16 melanoma and rapidly induced tumor-specific CTLs. Radhakrishnan et al., J. Immunol. 170 : 1830-38 (2003); Radhakrishnan et al., Cancer Res. 64 : 4965-72 (2004); Heckman et al., Eur. J. Immunol. 37 : 1827-35 (2007); (2) Blocks the development of airway inflammatory disease in a mouse allergic asthma model. Radhakrishnan et al., J. Immunol. 173 : 1360-65 (2004); Radhakrishnan et al., J. Allergy Clin. Immunol. 116 : 668-74 (2005).

Further evidence for reverse signaling to dendritic cells ("DCs") comes from studies in which bone marrow-derived DCs were cultured with soluble PD-1 (the PD-1 EC domain fused to an Ig constant region—"s-PD-1"). Kuipers et al., Eur. J. Immunol. 36 :2472-82 (2006). This sPD-1 inhibited DC activation and increased IL-10 production in a manner that could be reversed by administration of anti-PD-1.

In addition, several studies have shown receptors for PD-L1 or PD-L2 that are independent of PD-1. B7.1 has been identified as a binding partner of PD-L1. Butte et al., Immunity 27 : 111-22 (2007). Chemical cross-linking studies have shown that PD-L1 and B7.1 can interact through their IgV-like domains. B7.1: PD-L1 interactions can induce inhibitory signals in T cells. PD-L1 is connected by B7.1 on CD4+ T cells or B7.1 is connected by PD-L1 on CD4+ T cells to transmit inhibitory signals. When stimulated by anti-CD3 plus B7.1-coated beads, T cells lacking CD28 and CTLA-4 show reduced proliferation and cytokine production. In T cells lacking all receptors for B7.1 (i.e., CD28, CTLA-4, and PD-L1), T cell proliferation and cytokine production are no longer inhibited by anti-CD3 plus B7.1-coated beads. This suggests that B7.1 acts specifically through PD-L1 on T cells in the absence of CD28 and CTLA-4. Similarly, T cells lacking PD-1 showed reduced proliferation and cytokine production when stimulated in the presence of anti-CD3 plus PD-L1-coated beads, indicating an inhibitory effect of PD-L1 ligation on B7.1 on T cells. When T cells lack all known receptors for PD-L1 (i.e., no PD-1 and B7.1), T cell proliferation is no longer impaired by anti-CD3 plus PD-L1-coated beads. Therefore, PD-L1 can exert its inhibitory effect on T cells through B7.1 or PD-1.

The direct interaction between B7.1 and PD-L1 shows that the current understanding of co-stimulation is incomplete and underestimates the importance of the expression of these molecules to T cells. Studies on PD-L1 ^-/- T cells have shown that PD-L1 on T cells can downregulate the production of T cell cytokines. Latchman et al., Proc. Natl. Acad. Sci. USA (Proceedings of the National Academy of Sciences of the United States) 101 : 10691-96 (2004). Because both PD-L1 and B7.1 are expressed on T cells, B cells, DCs and macrophages, there may be a directional interaction between B7.1 and PD-L1 on these cell types. In addition, PD-L1 on non-hematopoietic cells can interact with B7.1 and PD-1 on T cells, raising the question of whether PD-L1 is involved in their regulation. One possible explanation for the inhibitory effect of the B7.1: PD-L1 interaction is that T cell PD-L1 can capture or isolate APC B7.1 from interacting with CD28.

Thus, antagonizing signaling through PD-L1, including blocking PD-L1 from interacting with PD-1, B7.1, or both, thereby preventing PD-L1 from sending negative co-stimulatory signals to T cells and other antigen-presenting cells, may enhance immunity in response to infection (e.g., acute and chronic) and tumor immunity. In addition, the anti-PD-L1 antibodies of the present invention can be combined with antagonists of other components of PD-1:PD-L1 signaling (e.g., antagonistic anti-PD-1 and anti-PD-L2 antibodies).

4. B7-H3

Costimulatory signals can also be provided by B7-H3 (B7RP-2, CD276, PRO352), which is widely expressed in lymphoid and non-lymphoid tissues. Chapoval et al., Nat. Immunol. 2 : 269-74 (2001). In humans, B7-H3 has 4Ig and 2Ig variants, with the 4Ig form being the predominant form and the 2Ig variant being the predominant form in mice. Sun et al., J. Immunol. 168 : 6294-97 (2002); Steinberger et al., J. Immunol. 172 : 2352-59 (2004); Ling et al., Genomics 82 : 365-77 (2003).

Recent studies have shown that B7-H3 is both a stimulator and an inhibitor of T cell responses. Evidence for stimulatory activation is provided by: (1) in combination with anti-CD3, B7-H3/Ig fusions costimulate the proliferation of CD4+ and CD8+ T cells and stimulate the lytic activity of IFN-γ and CD8, Chapoval et al., Nat. Immunol. 2 :269-74 (2001); and (2) injection of a B7-H3 expression plasmid into tumors in the EL-4 lymphoma model resulted in 50% complete tumor regression, which was dependent on CD8+ T cells and NK cells. However, several recent studies have shown an inhibitory effect of this molecule. B7-H3 ^-/- APC knockout showed a two-fold increase in alloreactive T cell proliferation in the MLR response. Activation of CD4 T cells by anti-CD3 and anti-CD28 was inhibited in HLA-DR2 transfected with either form of B7-H3. Ling et al., Genomics 82 : 365-77 (2003). The result was reduced proliferation and production of IFN-γ, TNF-α, IL-10, and GM-CSF. The reconciliation of these studies is that there are two functionally opposing receptors for B7-H3, similar to how CD28 and CTLA-4 regulate signaling through B7.1 and B7.2.

Therefore, blockade of B7-H3 signaling when combined with the anti-PD-L1 antibodies of the invention may promote enhanced immune responses to infection and tumor immunity.

5. B7-H4

A recent addition to the B7 family is B7-H4 (B7x, B7-S1, B7-H.5, VTCN1, PRO1291), a negative regulator of T cell responses. Zang et al., Proc. Natl. Acad. Sci. USA 100 (18), 10388-10392 (2003); Watanabe et al., Nat. Immunol. 4 (7), 670-679 (2003); Prasad et al., Immunity 18 (6), 863-873 (2003); Sica et al., Immunity 18 (6), 849-861 (2003). B7-H4 is widely expressed in both human and mouse lymphoid organs (spleen and thymus) and non-lymphoid organs (including lung, liver, testis, ovary, placenta, skeletal muscle, pancreas, and small intestine). Neither B7-H4 nor B7-H4 regulation at the translational level has been detected by IHC in normal human tissues. IHC shows high expression of B7-H4 in lung and ovarian tumors, and real-time polymerase chain reaction (PCR) analysis indicates that mouse B7-H4 is also highly expressed in prostate, lung, and colon cancer cell lines. B7-H4 binds to an as-yet-unidentified receptor on activated but not naive T cells, distinct from CTLA-4, ICOS, PD-1, and B7-H3 receptors. Although BTLA was initially reported to be a ligand for B7-H4, the reported binding of B7-H4/Ig fusions to wild-type but not BTLA ^-/ - cells has led to the conclusion that HVEM, rather than BTLA, is the unique ligand for B7-H4. Sedy et al., Nat. Immunol. 6 :90-98 (2004).

Studies with B7-H4 transfectants and immobilized B7-H4/Ig fusions demonstrate that B7-H4 transmits signals that inhibit TCR-mediated CD4 ⁺ and CD8 ⁺ T cell proliferation, cell cycle progression through the G0/G1 phase, and IL-2 production. Sica et al., Immunity 18 :849-61 (2003); Zang et al., PNAS 100 :10388-92 (2003); Prasad et al., Immunity 18 :863-73 (2003). B7.1 co-stimulation cannot overcome B7-H4/Ig-induced inhibition. Blocking anti-B7-H4 antibodies increase T cell proliferation and IL-2 production in vitro. In vivo administration of anti-B7-H4 antibodies comparable to keyhole limpet hemocyanin (KLH) in complete Freund's adjuvant (CFA) resulted in a moderate increase in anti-KLH antibody IgM production and a two- to three-fold increase in T cell proliferation and IL-2 production following in vitro restimulation with KLH, indicating greater T cell priming in the presence of anti-B7-H4 in vivo. Anti-B7-H4 blocking antibodies significantly accelerated the onset and severity of EAE, increasing CD4 ⁺ and CD8 ⁺ T cells and CD11b ⁺ macrophages in the brain of anti-B7-H4-treated autoimmune mouse models. Available combined experimental data on B7-H4 suggest that it can downregulate immune responses in peripheral tissues and play a role in mediating T cell tolerance. B7-H4 expression may also play a role in evading host immune responses in tumor immunity. Choi et al., J. Immunol. 171 :4650-54 (2003). Therefore, antagonizing B7-H4 when combined with the anti-PD-L1 antibodies of the invention can be used to enhance immune responses to infection and tumor immunity.

6. BTLA :

B7 family member BTLA (CD272, BTLA-1) has similar functions to PD-1 and CTLA. Initially identified as a selective marker for Th1 cells, BTLA is only expressed on lymphocytes. Similar to CTLA-4, ICOS and PD-1, BTLA is induced on T cells during activation. However, compared with ICOS (which remains increased on Th2 cells but downregulated in Th1 cells), BTLA remains expressed on Th1 cells, but not on Th2 cells. Similar to PD-1, BTLA is also expressed on B cells. Gavrieli et al., Biochem.Biophys.Res.Commun. (Biochemistry and Biophysics Research Communications) 312 : 1236-43 (2003). However, BTLA is expressed on both resting and activated B cells, while PD-1 is upregulated on activated B cells. BTLA has two ITIM motifs.

BTLA exerts an inhibitory effect on both B and T lymphocytes. Watanabe et al., Nat. Immunol. 4 : 670-79 (2003). BLTA ^-/- B cells showed a moderate response to anti-IgM, but showed an increased response to anti-CD3 in vitro. Polarized BTLA ^-/- Th1 cells showed an approximately two-fold increase in proliferation in response to in vitro antigen exposure. In vivo, BTLA ^-/- mice showed a three-fold increase in hapten-specific antibody responses and enhanced susceptibility to EAE. The phenotype of ^{BTLA -/-} mice is similar to that of PD-1 ^-/- mice, showing increased susceptibility to autoimmunity, but a more subtle phenotype than CTLA-4 ^-/- mice. However, considering its role as a negative regulator, blocking BTLA proves to be useful for enhancing immune responses in infection and anti-tumor immunity when combined with the anti-PD-L1 antibodies of the present invention.

Interestingly, the Ig superfamily member BTLA has recently been shown to interact with the TNFR family member HVEM. Sedy et al., Nat. Immunol. 6 :90-98 (2005); Gonzalez et al., Proc. Natl. Acad. Sci. USA 102 :1116-1121 (2005). HVEM is reviewed below under TNFR family co-stimulators.

E. TNFR family co-stimulatory factors

1.OX40/OX40L(CD134)

Mice deficient in OX40 (CD134, TXPG1L, TNFRSF4) and OX40L (CD134L, CD252, GP34, TNFSF4, TXGP1) have reduced primary CD4+ T cell responses to both viral and common protein antigens and in contact-sensitivity reactions. Chen et al., Immunity 11 : 689-698 (1999); Kopf et al., Immunity 11 : 699-708 (1999); Murata et al., J. Exp. Med. 191 : 365-374 (2000); Gramaglia et al., J. Immunol. 165 : 3043-3050 (2000). Lower frequencies of antigen-specific effector T cells and less memory T cell development occur in the late stages of the primary response. Gramaglia et al., supra. Compared to CD27-deficient T cells, early proliferation is not impaired in naive CD4+ T cell populations with OX40 deletion. However, reduced proliferation and significant apoptotic cell death occur 4-5 days after activation, resulting in few T cells surviving long-term. Rogers et al., Immunity 15 : 445-455 (2001). For OX40-deficient CD8+ T cells, initial cell division is unaffected, but the accumulation of primary effector cells is significantly reduced 3-6 days after encountering antigen. Croft et al., Nat. Immunol. 3 : 609-620 (2003).

Transgenic expression of OX40L by dendritic cells or T cells increases the number of antigen-responsive CD4+ T cells, resulting in autoimmune-like symptoms associated with abnormal T cell activation. Brocker et al., Eur. J. Immunol. 29 :1610-1616 (1999); Murata et al., J. Immunol. 169 :4628-4636 (2002). Following immunization, injection of agonistic anti-OX40 antibodies leads to the accumulation of greater numbers of antigen-reactive CD4+ T cells at the peak of the primary response, accompanied by an increase in the number of memory T cells generated. Gramaglia et al., supra., Bansai-Pakala et al., Nature Med. 7 :907-912 (2001), Maxwell et al., J. Immunol. 164 :107-112 (2000); Weatherill et al., Cell. Immunol. 209 :63-75 (2001). Increased accumulation of primary effector CTLs occurs when antigen-sensitized mice are treated with agonistic antibodies specific for OX40. De Smedt et al., J. Immunol. 168 :661-670 (2002).

OX40 is thought to provide a late-acting signal that allows the survival of newly generated effector cells at the peak of the primary immune response. There is also good evidence that OX40 functions downstream of CD28 - in addition to increasing the expression of OX40 mediated by CD28 signaling, functional analysis of CD28 deficiency relative to OX40 deficiency has shown that early primary T cell responses are significantly impaired in the absence of CD28 signaling, but only late responses are impaired in the absence of OX40 signaling. Rogers et al., Immunity 15 : 445-455 (2001); Bertram et al., J. Immunol. 168 : 3777-3785 (2002).

Therefore, activation of OX40/OX40L (such as by use of agonistic antibodies) when combined with the anti-PD-L1 antibodies of the invention may be useful in treating T cell dysfunction disorders.

2.4-1BB(CD137)/4-1BBL,

Similar to OX40/OX40L, T cells lacking 4-1BB (CD137, TNFRSF9) and 4-1BBL (TNFSF9) show less antigen-reactive CD8+T cell accumulation and less memory T cell development in the primary response when there is no 4-1BBL. DeBenedette et al., J.Immunol. (Journal of Immunology) 163 : 4833-4841 (1999); Tan et al., J.Immunol. (Journal of Immunology) 163 : 4859-4868 (1999); Tan et al., J.Immunol. (Journal of Immunology) 164 : 2320-2325 (2000). In addition, blocking 4-1BBL does not change the initial proliferation response of CD8+T cells, but suppresses the accumulation of effector CTLs at the peak of the primary response after 3-6 days, which is due to the apoptosis of cells that have divided several times. Cooper et al., Eur. J. Immunol. 32 : 521-529 (2002). Similar results were also produced by agonistic anti-4-1BB antibodies and anti-4-1BBL-transfected APCs: CTL and CD4+ T cell responses in vivo were significantly increased. Melero et al., Nature Med. 3 :682-685 (1997); Melero et al., Eur. J. Immunol. 28 :1116-1121 (1998); Takahashi et al., J. Immunol. 162 :5037-5040 (1999); Guinn et al., J. Immunol. 162 :5003-5010 (1999); Halstead et al., Nature Immunol. 3 :536-541 (2002); Takahashi et al., Immunol. Lett. 76 :183-191 (2001); Bansal-Pakala et al., J. Immunol. 169: :5005-5009 (2002).4-1BB-specific antibodies do not change the initial proliferation response, supporting the conclusion of the 4-1BBL blocking experiment, indicating that 4-1BB is active in the late stage of providing cell survival signals.

Similar to OX40, it is believed that 4-1BB provides a late action signal, which allows the survival of newly generated effector cells at the peak of the primary immune response. There is also good evidence that 4-1BB functions later than CD28 - in addition to the increased expression of OX40 and 4-1BB mediated by CD28 signals, functional analysis of CD28 deletions versus 4-1BB deletions has been shown to significantly damage early primary T cell responses in the absence of CD28 signals, but only late responses are destroyed in the absence of OX40 signals. Rogers et al., Immunity (immunity) 15 : 445-455 (2001); Bertram et al., J. Immunol. (immunology journal) 168 : 3777-3785 (2002).

Agonistic anti-CD137 antibodies can induce tumor regression in cancer, with CD8+ CTLs playing a central role. Melero et al., Nat. Med. 3 :682-5 (1997); Hirano et al., Cancer Res. 65 (3):1089-96 (2005). Constitutive and inducible PD-L1 expression in such tumors confers resistance, which is reversible upon PD-L1 blockade. Hirano et al.

Therefore, activation of 4-1BB/4-BBL (such as by the use of agonistic antibodies), particularly in combination with PD-L1 antagonists (e.g., anti-PD-L1 antibodies), may be useful in treating T cell dysfunction disorders.

3.CD27/CD27L (CD70)

The importance of CD27 (TNFRSF7, S152) and CD27L (CD70, TNFSF7) signaling in the initial stages of T cell responses has been demonstrated in in vitro blocking studies in which the CD27/CD70 interaction was disrupted. Oshima et al., Int. Immunol. 10 :517-526 (1998); Agematsu et al., J. Immunol. 153 :1421-1429 (1994); Hintzen et al., J. Immunol. 154 :2612-2623 (1995). T cells lacking CD27 initially divide normally but then proliferate poorly for 3 or more days after activation. Hendriks et al., Nature Immunol. 1 :433-440 (2000). This suggests that CD27 is involved in promoting the initial expansion of naive T cell populations, either by inhibiting T cell death early or by acting on the cell cycle to allow for continued division 2-3 days after activation. This is reinforced by in vivo studies in CD27-deficient mice, in which fewer antigen-specific responses (days 4-8) and fewer memory T cells develop over a period of 3 weeks or more. Hendriks et al., supra. Early CD27 expression is upregulated after T cell activation, suggesting that it primarily transmits signals that maintain early proliferation before the peak of the effector response.

Thus, activation of CD27/CD27L, including activation through the use of agonistic antibodies, particularly in combination with the anti-PD-L1 antibodies described herein, may be useful in treating T cell dysfunction disorders.

4.CD30/CD30L (CD153)

CD30 (TNFRSF8, Ki-1) and CD30L (CD153, TNFSF8) signaling are costimulatory for several T cell functions in vitro. Del Prete et al., J. Exp. Med. 182 :1655-1661 (1995), Bowen et al., J. Immunol. 156 :442-449 (1995). Blocking agents for CD30L inhibit Th2 cell development and enhance Th1 cell development in vitro. This activity is consistent with data showing that CD30 is preferentially expressed by Th2 cells and type 2 cytotoxic T cells. Del Prete et al., supra, Nakamura et al., J. Immunol. 158 :2090-2098 (1996). In unpolarized primary responses, CD30 is expressed 3-4 days after activation of naive T cells. Nakamura et al., supra, showed that its function is not restricted to type 2 cytokine-dominated responses.

Although the precise mechanism of CD30/CD30L signaling is unclear, it has been suggested that it may be similar to OX40 and 4-1BB. When adoptively transferred antigen-specific CD8+ T cells are transferred into CD30L-deficient mice, they do not accumulate in large numbers at the peak of the primary response, and fewer memory T cells develop. Therefore, CD30 may also provide proliferation and/or survival signals to allow the generation of large numbers of antigen-specific T cells at the peak of the primary response.

Thus, activation of CD27/CD27L (including through the use of agonistic antibodies), particularly in combination with the anti-PD-L1 antibodies described herein, may be useful in treating T cell dysfunction disorders.

5.HVEM/LIGHT

The effects of HVEM (HVEA, ATAR, LIGHTR, TNFRSF14, PRO509) and LIGHT (CD258, HVEML, TR2, TNFSF14, PRO726) on T cell costimulation are complicated by: 1) the ability of LIGHT to also bind to the lymphotoxin-β receptor (LTβR), and 2) HVEM binding to soluble LTα3. Therefore, any study of HVEM/LIGHT effects should consider the effects on other binding partners of this signaling system. Blocking LIGHT inhibits early T cell proliferation and cytokine secretion in allogeneic mixed-lymphocyte reactions (MLRs). Tamada et al., J. Immunol. 164 : 4105-4110 (2000), Kwon et al., J. Biol. Chem. 272 : 14272-14276 (1997); Harrop et al., J. Immunol. 161 : 1786-1794 (1998); Tamada et al., Nature Med. 6 : 283-289 (2000). When LIGHT is blocked in MHC-mismatched cardiac allografts, the production of proinflammatory cytokines is suppressed. Ye et al., J. Exp. Med. 195 : 795-800 (2002). In addition, allogeneic skin grafts are rejected with delayed kinetics in recipients lacking both LIGHT and CD28. Scheu et al., J. Exp. Med. 195 : 1613-1624 (2002). It has been proposed that delayed graft rejection may indicate an early inhibition of T cell clonal expansion or cytokine production. This conclusion is supported by (i) in vitro studies showing that LIGHT-deficient splenocytes responding to alloantigens have reduced production of both TH1 and TH2 cytokines and weak cytotoxic T lymphocyte (CTL) activity [Sheu et al., supra.], and (ii) in vivo studies showing that blocking LIGHT reduces the production of alloreactive CTLs. Tamada et al., Nature Med. 6 : 283-289 (2000).

Thus, HVEM/LIGHT (such as by use of agonistic antibodies), particularly in combination with the anti-PD-L1 antibodies described herein, can be used to treat T cell dysfunction disorders.

II. Definitions

"Allergen" or "immunogen" is any molecule that can trigger an immune response. When used herein, the term covers the antigenic molecule itself or its source, such as pollen grains, animal dander, insect venom or food. This is compared to the term antigen, which refers to a molecule that can be specifically recognized by an immunoglobulin or T cell receptor. Any foreign substance that can induce an immune response is a potential allergen. Many different chemicals of natural and synthetic origin are known to be allergenic. Complex natural organic chemicals (particularly proteins) may cause antibody-mediated allergic reactions, while simple organic compounds, inorganic chemicals and metals more preferably cause T cell-mediated allergic reactions. In some cases, the same allergen can cause more than one type of allergic reaction. Exposure to the allergen can be by inhalation, injection, injection or skin contact.

In the context of immune dysfunction, "dysfunction" refers to a state of reduced immune response to antigenic stimulation. The term encompasses elements common to both exhaustion and/or anergy, in which antigen recognition occurs but the ensuing immune response is ineffective in controlling infection or tumor growth.

"Tolerance" or "immune tolerance" is the inability of the immune system to initiate a defensive immune response to a specific antigen. Tolerance can be natural or autologous, in which the body does not attack its own proteins and antigens, or tolerance can be induced, resulting from manipulation of the immune system. Central tolerance occurs during lymphocyte development, taking place in the thymus and bone marrow. In this process, T and B lymphocytes that recognize self-antigens are eliminated before they develop into fully immunocompetent cells. This process is most active during fetal development, but continues throughout life, producing immature lymphocytes. Peripheral T cell tolerance refers to functional unresponsiveness to self-antigens present in peripheral tissues, which occurs after T and B cells mature and enter the periphery. These processes include the suppression of autoreactive cells by "regulatory" T cells and the generation of hyporesponsiveness (anergy) in lymphocytes that encounter antigens in the absence of co-stimulatory signals associated with inflammation. "Acquired" or "induced tolerance" refers to the adaptation of the immune system to external antigens, characterized by specific unresponsiveness of lymphoid tissues to a given antigen, for which cell-mediated or humoral immunity would otherwise be induced. In adults, tolerance can be induced clinically by repeated administration of large doses of antigen or small doses below the threshold required to stimulate an immune response, such as by intravenous or sublingual administration of soluble antigen. Immunosuppression also assists in the induction of tolerance. Breakdown of self-tolerance can lead to autoimmunity.

"Enhancing T cell function" means inducing, causing or stimulating T cells to have a sustained or expanded biological function, or restoring or reactivating exhausted or inactivated T cells. Examples of enhancing T cell function include: increasing the secretion of gamma interferon from CD8 ⁺ T cells relative to such levels before intervention, increasing proliferation, increasing antigen responsiveness (e.g., virus or pathogen clearance). In one embodiment, the level of enhancement is at least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. The manner of measuring this enhancement is known to those of ordinary skill in the art.

A "T cell dysfunction disorder" is a disorder or condition of T cells characterized by decreased responsiveness to antigenic stimulation. In a specific embodiment, a T cell dysfunction disorder is a disorder specifically associated with inappropriately increased signaling through PD-1. In another embodiment, a T cell dysfunction disorder is a disorder in which T cells are anergic or have a decreased ability to secrete cytokines, proliferate, or perform cytolytic activities. In a specific aspect, the decreased responsiveness results in ineffective control of pathogens or tumors expressing immunogens. Examples of T cell dysfunction disorders characterized by T cell dysfunction include unresolved acute infections, chronic infections, and tumor immunity.

" Chronic infection " refers to such infection, wherein infectious agent (for example, pathogen such as virus, bacterium, protozoan parasite, fungus or the like) has induced immune response in the host of infection, but has not yet been cleared or eliminated from the host as in acute infection process. Chronic infection can be persistent, latent or slow. Although acute infection is typically resolved by the immune system in a few days or weeks (such as influenza), persistent infection can last for months, years, decades or a lifetime (for example, hepatitis B) at a relatively low level. In contrast, the feature of latent infection is long-term asymptomatic activity, interrupted from time to time by a period of rapid increase in high infection and elevated pathogen levels (for example herpes simplex). Finally, the feature of slow infection is the gradual and continuous increase of disease symptoms, such as long-term incubation period, followed by the start of a prolonged and progressive clinical course after clinical symptoms appear. Unlike latent and persistent infections, chronic infections may not begin with an acute phase of viral proliferation (e.g., picornavirus infections, visna virus, scrapie, Creutzfeldt-Jakob disease). Exemplary infectious agents capable of inducing chronic infection include viruses (e.g., cytomegalovirus, Epstein-Barr virus, hepatitis B virus, hepatitis C virus, herpes simplex virus types 1 and 2, human immunodeficiency virus types 1 and 2, human papillomavirus, human T-lymphotropic virus types 1 and 2, varicella-zoster virus, etc.), bacteria (e.g., Mycobacterium tuberculosis, Listeria spp., Klebsiella pneumoniae, Streptococcus pneumoniae, Staphylococcus aureus, Borrelia spp., Helicobacter pylori, etc.), protozoan parasites (e.g., Leishmania spp., Plasmodium falciparum, Schistosoma spp.), spp.), Toxoplasma spp., Trypanosoma spp., Taenia carssiceps, etc.), and fungi (e.g., Aspergillus spp., Candida albicans, Coccidioides immitis, Histoplasma capsulatum, Pneumocystis carinii, etc.). Additional infectious agents include prions or misfolded proteins that affect brain or neuronal structures by further propagating protein misfolding in these tissues, leading to the formation of amyloid plaques (which lead to cell death, tissue damage, and ultimately death). Examples of diseases caused by prion infection include: Creutzfeldt-Jakob disease and its varieties, Gerstmann--Scheinker syndrome (GSS), fatal familial insomnia (sFI), kuru, scrapie, bovine spongiform encephalopathy (BSE) in cattle (aka "mad cow" disease), and various other animal forms of encephalopathy [e.g., transmissible mink encephalopathy (TME)], chronic wasting disease (CWD) in white-tailed deer, elk, and mule deer, feline spongiform encephalopathy (FSE), and feline spongiform encephalopathy (FSE). encephalopathy, exotic ungulate encephalopathy (EUE) in nyala, oryx, and greater kudu, spongiform encephalopathy of the ostrich].

"Tumor immunity" refers to the process by which tumors evade immune recognition and clearance. Therefore, as a therapeutic concept, tumor immunity is "treated" when this evasion is reduced, allowing the tumor to be recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

"B7-negative co-stimulatory antagonists" ("BNCA") are agents that reduce, block, inhibit, eliminate, or interfere with negative co-stimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes that are mediated by members of the B7 family. In one aspect, BNCA can render dysfunctional T cells non-dysfunctional, alone or in combination with the anti-PD-1 antibodies of the present invention. In another aspect, BNCA can be an agent that inhibits nucleic acid or protein synthesis, expression, signal transduction, and/or post-expression processing of a B7-negative co-stimulatory molecule. In yet another aspect, BNCA is an antibody, an antigen-binding antibody fragment, a BNCA oligopeptide, a BNCA RNAi, or a BNCA small molecule that reduces, blocks, inhibits, eliminates, or interferes with signal transduction of a B7-negative co-stimulatory molecule. Exemplary B7 negative co-stimulatory molecules include: CTLA-4, PD-L1, PD-1, B7.1 (expressed on T cells), PD-L2, B7-H3, and B7-H4.

Positive costimulatory agonists are molecules that increase, enhance, promote or assist in the costimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes. On the one hand, positive costimulatory molecules can be extracellular domains, soluble constructs or agonistic antibodies that activate positive costimulatory pathways. Exemplary positive costimulatory molecules include B7 superfamily molecules, for example, B7.1, B7.2, CD28 and ICOS/ICOSL. Other examples include TNFR family costimulatory molecules, for example, OX40/OX40L, 41-BB/41-BBL, CD27/CD27L, CD30/CD30L and HVEM/LIGHT.

A "small molecule" or "small organic molecule" is a molecule having a molecular weight of less than about 500 Daltons.

"Interfering RNA" or "RNAi" is an RNA that reduces the expression of a target gene of 10-50 nucleotides in length, wherein a portion of the chain is sufficiently complementary (e.g., having at least 80% identity to the target gene). The method of RNA interference refers to target-specific inhibition of gene expression (i.e., "gene silencing"), which occurs at the post-transcriptional level (e.g., translation), and includes all post-transcriptional and transcriptional mechanisms of RNA-mediated inhibition of gene expression, such as those described below: P.D. Zamore, Science 296 : 1265 (2002) and Hannan and Rossi, Nature 431 : 371-378 (2004). When used herein, RNAi can be in the form of small interfering RNA (siRNA), short hairpin RNA (shRNA), and/or microRNA (miRNA). Such RNAi molecules are often double-stranded RNA complexes that can be expressed in the form of separated complementary or partially complementary RNA chains. Methods for designing double-stranded RNA complexes are known in the art. For example, the design and synthesis of suitable shRNAs and siRNAs can be found in Sandy et al., BioTechniques 39 :215-224 (2005).

"Small interfering RNA" or siRNA is a double-stranded RNA (dsRNA) duplex of 10-50 nucleotides in length that reduces the expression of a target gene, wherein a portion of the first strand is sufficiently complementary (e.g., at least 80% identical to the target gene). siRNAs are specifically designed to avoid antiviral responses characterized by increased interferon synthesis, nonspecific protein synthesis inhibition, and RNA degradation, which often lead to cell suicide or death in mammalian cells associated with the use of RNAi. Paddison et al., Proc Natl Acad Sci USA 99 (3): 1443-8. (2002).

The term "hairpin" refers to a circular RNA structure of 7-20 nucleotides. "Short hairpin RNA" or shRNA is a single-stranded RNA of 10-50 nucleotides in length characterized by a hairpin bend that reduces target gene expression, wherein the RNA strand portion is sufficiently complementary (e.g., at least 80% identical to the target gene). The term "stem-loop" refers to the pairing between two regions of base pairs in the same molecule to form a double helix terminated by a short unpaired loop, resulting in a lollipop-shaped structure.

"MicroRNAs" or "miRNAs" (formerly known as stRNAs) are single-stranded RNAs of approximately 10-70 nucleotides in length that are initially transcribed as pre-miRNAs characterized by a "stem-loop" structure, which are subsequently processed into mature miRNAs after further processing by the RNA-induced silencing complex (RISC).

"BNCA interfering RNA" or "BNCA RNAi" binds (preferably specifically binds) to a BNCA nucleic acid and reduces its expression. This means that expression of the B7 negative co-stimulatory molecule is lower in the presence of BNCA RNAi compared to expression of the B7 negative co-stimulatory molecule in a control (in which BNCA RNAi is absent). BNCA RNAi can be identified and synthesized using known methods (Shi Y., Trends in Genetics 19 (1): 9-12 (2003), WO2003056012, WO2003064621, WO2001/075164, WO2002/044321).

"BNCA oligopeptides" are oligopeptides that bind, preferably specifically bind, to a B7 negative co-stimulatory polypeptide, including a receptor, ligand, or signaling component, respectively, as described herein. Such oligopeptides can be chemically synthesized using known oligopeptide synthesis methods or can be prepared and purified using recombinant technology. Such oligopeptides are typically at least about 5 amino acids long, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 98, 99, or 100 amino acids in length or longer. In this regard, it is noted that techniques for screening oligopeptide libraries for oligopeptides capable of specifically binding to a polypeptide target are well known in the art (see, e.g., U.S. Patent Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506 and WO 84/03564; Geysen et al., Proc. Natl. Acad. Sci. USA, 81 :3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. USA, 82 :178-182 (1985); Geysen et al., in Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J. Immunol. Meth., 102 : 259-274 (1987); Schoofs et al., J. Immunol. (Journal of Immunology), 140 : 611-616 (1988), Cwirla, SE et al., Proc. Natl. Acad. Sci. USA (Proceedings of the National Academy of Sciences of the United States of America), 87 : 6378 (1990); Lowman, HB et al., Biochemistry, 30 : 10832 (1991); Clackson, T. et al., Nature, 352 : 624 (1991); Marks, JD et al., J. Mol. Biol., 222 :581 (1991); Kang, AS et al. Proc. Natl. Acad. Sci. USA (Proceedings of the National Academy of Sciences of the United States of America), 88 :8363 (1991), and Smith, GP, Current Opin. Biotechnol., 2 :668 (1991).

" BNCA small molecule antagonist " or " BNCA small molecule " is an organic molecule other than an oligopeptide or antibody as defined herein, which inhibits (preferably specifically inhibits) a B7 negative costimulatory polypeptide. Such B7 negative costimulatory signaling inhibition preferably causes dysfunctional T cells to respond to antigen stimulation. Examples of BNCA small molecules can be identified and chemically synthesized using known methods (see, e.g., PCT Publication Nos. WO2000/00823 and WO2000/39585). Such BNCA small molecules are generally less than about 2000 daltons in size, alternatively less than about 1500,750,500,250 or 200 daltons in size, capable of binding (preferably specifically binding) a B7 negative stimulatory polypeptide described herein, and can be identified without excessive experimentation using well-known technology. In this regard, it is noted that the technology for screening organic molecule libraries for obtaining molecules capable of binding to polypeptide targets is well known in the art (see, e.g., PCT Publication Nos. WO00/00823 and WO00/39585).

The term "antibiotic" is included in any molecule that specifically inhibits or eliminates microbial growth at an applied concentration and dosing interval, but is not lethal to the host, such as viruses, bacteria, fungi, or protozoa. When used herein, the term antibiotic includes antimicrobial agents, antivirals, antifungals, and antiprotozoal agents. In a specific aspect, antibiotics are nontoxic to the host at an applied concentration and dosing interval. Antibacterial antibiotics or antimicrobials can be broadly classified as bactericidal (i.e., directly killing) or bacteriostatic (i.e., preventing division). Antibacterial antibiotics can be further reclassified as narrow spectrum (i.e., only affecting small bacterial subtypes, for example, Gram-negative, etc.) or broad spectrum (i.e., affecting a wide range of species). Examples of antibiotics include: (i) aminoglycosides, e.g., amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, paromycin, (ii) ansamycins, e.g., geldanamycin, herbimycin, (iii) carbacephems, e.g., loracarbef, (iv) carbapenems, e.g., ertapenum, doripenem, imipenem/cilastatin, meropenem, (v) cephalosporins (first generation), for example, cefadroxil, cefazolin, cefalotin, cefalexin, (vi) cephalosporins (second generation), for example, ceflaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, (vi) cephalosporins (third generation), for example, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, (vii) cephalosporins (fourth generation), for example, cefepime; (viii) cephalosporins (fifth generation), for example, ceftobiprole; (ix) glycopeptides, for example, teicoplanin, vancomycin; (x) macrolides, for example, axithromycin, clarithromycin, dirithromycine, erythromycin, roxithromycin, troleandomycin, telithromycin, spectinomycin; (i) penicillins, for example, amoxicillin, ampicillin, axlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcilin, oxacillin, penicillin, peperacillin, ticarcillin, (xiii) antibiotic polypeptides, for example, bacitracin, colistin, polymyxin B, B), (xiv) quinolones, for example, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lemefloxacin, moxifloxacin, norfloxacin, orfloxacin, trovafloxacin, (xv) sulfonamides, for example, mafenide, prontosil, sulfacetamide, sulfamethizole, sulfanilamide, sulfasalazine, sulfisoxazole, soxazole), trimethoprim, trimethoprim-sulfamethoxazole (TMP-SMX), (xvi) tetracyclines, for example, demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, and (xvii) others such as arspenamine, chloramphenicol, clindamycin, lincomycin, ethambutol, fosfomycin, fusidic acid, acid, furazolidone, isoniazid, linezolid, metronidazole, mupirocin, nitrofurantoin, platensimycin, pyrazinamide, quinupristin/dalfopristin, rifampin/rifampicin, or tinidazole.

The term "antiviral agent" includes any molecule that inhibits or eliminates viral growth, morbity and/or survival. This includes antiretroviral drugs such as (1) reverse transcriptase inhibitors including, for example: (a) nucleoside analog reverse transcriptase inhibitors (NRTIs) (e.g., acyclovir/acyclovir, cidofovir, azidothymidine/zidovudine (AZT,), didanosine (ddI,); zalcitabine (ddC,); stavudine (d4T,); lamivudine (3TC,); abacavir; emtricitabine; brivudine; entecavir; idoxuridine; viramidi ne (taribavirin, produced by Valeant Pharmaceuticals), cytidine analog polymerase inhibitor PCI-6130 and prodrug variants (e.g., R7128), produced by Pharmasset/Roche; nucleoside analog inhibitors, produced by Merck/Isis Pharmaceuticals – MK-0608, (b) nucleotide analog reverse transcriptase inhibitors (NtRTIs) such as tenofovir, adefovir, and fomivirsen; (c) non-nucleoside reverse transcriptase inhibitors (NNRTIs) such as efavirenz, nevirapine, delavirdine, and etravirine; and HCV Non-nucleoside inhibitors of RNA-dependent RNA polymerase from ViroChem Pharma – VCH-759, non-nucleoside inhibitors of HCV polymerase from Pfizer – PF-868554; and (d) polymerase inhibitors, including RNA-dependent RNA polymerase of hepatitis C virus from Boehringer Ingelheim – BILB-1941, RNA polymerase inhibitor from Roche – R1626; ACH-0137171, replicase inhibitor from Achillion Pharmaceuticals, R7128 – polymerase inhibitor from Roche/Pharmasset, ABT-333, and ABT-072 – polymerase inhibitors from Abbott, BI 207127 – polymerase inhibitor from Boehringer Ingelheim, PSI-7851 – a polymerase inhibitor, produced by Pharmasset, ANA598 – a polymerase inhibitor, produced by Anadys Pharmaceuticals, MK-3281 – a polymerase inhibitor, produced by Merck, IDX184 – a polymerase inhibitor, produced by Idenix, GSK 625433 – a polymerase inhibitor, produced by Glaxo Smith Kline, INX-189 – a polymerase inhibitor, produced by Inhibitex, NM283 – a polymerase inhibitor, produced by Idenix, HCV796 – a polymerase inhibitor, produced by Wyeth, GL60667 and GS9190 – polymerase inhibitors, produced by Gilead, PF-00868554 0 polymerase inhibitors, produced by Pfizer, VCH759, VCH916, VX222 and VX759 – polymerase inhibitors, produced by Virochem, IDX184 and IDX375 – polymerase inhibitors, produced by Idenix, BMS650032 – polymerase inhibitor, produced by Bristol Myers Squibb; (2) protease inhibitors include, for example: saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir, atazanavir, fosamprenavir, tipranavir, darunavirtelapravir (VX-950); second generation HCV protease inhibitors, produced by Vertex Pharmaceuticals – VX-500 and VX-813; NS3/4A protease inhibitors from Intermune/Roche – ITMN-191/R-7227, boceprevir, a protease inhibitor from Schering-Plough – SCH 503034, HCV NS3/4A protease inhibitors from Medivir/Tibotec – TMC435/TMC435350, ACH-1625 protease inhibitor from Achillion Pharmaceuticals, ACH-806 – a protease inhibitor from Achillion/Gilead, BI201335 and BILN2061 – protease inhibitors from Boehringer Ingelheim, SCH 900518/SP900518 (narlaprevir) – a protease inhibitor from Schering-Plough, MK-7009 – a protease inhibitor from Merck, BMS-650032, BMS-790052, and BMS-791325 – protease inhibitors from Bristol Myers Squibb, R7227 – a protease inhibitor from Roche, PHX1766 – a protease inhibitor from Phenomix, AVL-181 – a protease inhibitor from Avila Therapeutics, biliverdin, CTS-1027 – a protease inhibitor from Roche Biosciences, VX985 – protease inhibitor, produced by Vertex, VCH-759 and VCH-917 – protease inhibitors, produced by Virochem/Vertex, IDX-136 and 316 – protease inhibitors, produced by Idenix, ABT-450 – protease inhibitor, produced by Abbott, VBY 376 – protease inhibitors, produced by Virobay; (3) integrase inhibitors including, for example, raltegravir and elvitegravir; (4) combination therapy of nucleoside analog/nucleotide analog inhibitors, atripla (tenofovir + embricitabine + efavirenz), combivir (lamivudein + zidovudine), (5) entry or fusion inhibitors including, for example, maraviroc, enfuvirtide, docosanol, anti-CD4 antibodies, anti-gp120 antibodies, anti-CCR5 antibodies, HCV NS5a antagonists: (a) A-831, A-689 and AZD 2836, produced by Arrow Therapeutics, (b) BMS-790052 and BMS-824393, produced by Bristol Myers Squibb, (c) GSK-625433, produced by Glaxo Smith Kline, (d) NS4a antagonist ACH-1095; (5) maturation inhibitors including, for example, bevirimat and vivecon; (6) viral release inhibitors including, for example, zanamivir, oseltamivir, arbidol; (7) immune response enhancers including, for example, interferons - e.g., BLX-883 and BLX 883CR, produced by Biolex Therapeutics, belerofon, produced by Nautilus Biotech, long-acting IFN-α, IFN-α SR, produced by LG Life Sciences, long-acting IFN-α2b CR and IFN-α2b XL, produced by Flamel Technologies, pegylated IFN-α (e.g., PEG-IFN-α-2a, PEG-IFN-α-2b), IFN-α2b-human serum albumin fusion proteins, interferon-β including IFN-β-1b, interferon-γ, interferon-λ, pegylated interferon-λ (e.g., PEG-rIL-29, from ZymoGenetics/NovoNordisk), interferon-ω/leukocyte II interferon (e.g., Intarcia Therapeutics), toll-like receptor 7 agonists including imiquimod, isatoribine and its prodrug variants (e.g., ANA-975 and ANA-971), from Anadys Pharmaceuticals, oglufanide (IM862, L-Glu-L-Trp-OH) and its lipid- or glycosyl-conjugated variants, from Implicit Bioscience, NOV-205 (e.g., a peptide antiviral agent, produced by Novelos Therapeutics, Inc.), antiviral agent EHC18, produced by Enzo Biochem, γ-D-glutamyl-L-tryptophan (e.g., SCV-07, SciClone Pharmaceuticals/Verta), aloferon (e.g., aloferon-1-HGVSGHGQHGVHG, aloferon-2-GVSGHGQHGVHG), CPG 10101 – a TLR-9 agonist, produced by Coley Pharmaceuticals/Actilon; (8) antiviral synergist enhancers, i.e., agents that have little or no antiviral properties alone but enhance the effects of other antiviral agents, – e.g., choroquine, grapefruit juice, hydroxyurea, leflunomide, mycophenolic acid acid), resveratrol, ritonavir; and other antiviral drugs such as amantadine, edoxudine, famciclovir, penciclovir, fascarnet, fosfonet, ganciclovir, gardasil, ibacitabine, imunovir, moroxydine, nexavir, peramivir, pleconaril, podophyllotoxin, ribavirin, rimantadine, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vidarabine, and interferon-boosting agents such as EMZ702, produced by Transition Therapeutics, histamine dihydrochloride (e.g., +IFN-α); and (9) various or unclassified antiviral agents such as: KPE-02003002 (Artenimol), produced by Kemin Pharmaceuticals, mitoquinone-coenzyme Q10 antioxidant agonists, produced by Antipodean Pharmaceuticals, α-glucosidase I inhibitors (e.g., MX-3253-celgosivir, produced by Migenix Pharmaceuticals), castanospermine, glucocorticoid antagonists (e.g., HCV IRES inhibitor, mifepristone, VGX-410C, produced by VGX Pharmaceuticals), hepatic agonists (e.g., PYN17, produced by Phynova Pharmaceuticals), anti-viral agents derived from traditional herbal remedies, e.g., PYN18, produced by Phynova Pharmaceuticals Pharmaceuticals, caspase inhibitors (e.g., LB-84451 from LG LifeSciences, emricasan-PF-03491390/IDN-6556 from Pfizer), cyclosporine analogs that inhibit viral replication by preventing binding to cyclophilin A (e.g., SDZ NIM911 from Novartis, Debio-025 from Debiopharm).

The term "anti-fungal agent" includes any molecule that inhibits or eliminates the growth, morbity and/or survival of fungi. This includes, for example, (1) polyene antifungals such as natamyin, rimocidin, filipin, nystatin, amphotericin B, candicin; (2) imidazoles such as miconazole, ketoconazole, econazole, bifonazole, butoconazole, ketoconazole, fenticonazole, isoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole; (3) triazoles such as oxiconazole, sertaconazole, sulconazole, tioconazole; (4) oxiconazole, sertaconazole, sertaconazole, sulconazole, tioconazole; (5) oxiconazole, sertaconazole, sertaconazole, sertaconazole, sulconazole, tioconazole; (6) oxiconazole, sertaconazole, sertaconazole, sertaconazole, sulconazole, tioconazole; (7) oxiconazole, sertaconazole, sertaconazole, sertaconazole, sulconazole, tioconazole; (8) oxiconazole, sertaconazole, sertaconazole, sertaconazole, sulconazole, tioconazole; (9) oxiconazole, sertaconazole, sertaconazole, sertaconazole, sulconazole, tioconazole; (10) oxiconazole, sertaconazole, sertaconazole, sertaconazole, sulconazole, tioconazole; (11) oxiconazole, sertaconazole, sertaconazole, sertaconazole, sulconazole, tioconazole; (12) oxiconazole, sertaconazole, sertaconazole, sertacon ) such as fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole, voriconazole, terconazole; (4) allylamines such as terbinafine, amorolfine, naftifine cream, butenafine (LOTRIMIN); (5) echinocandins such as anidulafungin, caspofungin, micafungin and other substances with anti-fungal properties such as benzoic acid acid), cicclopix, flucytosine, griseofulvin, gentian violet, haloprogin, tolnaftate, undecylenic acid, tea tree oil (ISO 4730 (Oil of Melaleuca, terpinen-4-ol type), citronella oil, lemon grass, orange oil, palmarosa oil, patchouli, lemon myrtle, neem seed oil, and coconut oil.

The term "anti-protozoal agent" or "anti-protozoal agent" includes any molecule that inhibits or eliminates the growth, pathogenesis and/or survival of protozoal organisms. Examples of anti-protozoal agents include, (1) anti-malarial agents, for example, quinine, quinimax, quinidine, quinimax, chloroquine, Hydroxycloroquine, amodiaquine, pyrimethamine, sulphadoxine, proguanil, mefloquine, halofantrine, primaquine, artemesinin and its derivatives (e.g., artemether) (artemether, artensunate, dihydroartemisinin, arteether), clindamycin and combinations thereof; (2) protease inhibitors and drugs, benznidaole, buparvaquone, carbarsone, clioquinol, disulfiram, eflornithine, emetine, furazolidone, meglumine antimoniate, antimoniate, melarsoprol, metronidazole, miltefosine, nifurtimox, nitazoxanide, ornidazole, paromomycin sulfate, pentamidine, pyrimethamine, secnidazole, and tinidazole.

The term "vaccine" as used herein includes any non-pathogenic immunogen that, when inoculated into a host, induces protective immunity against a specific pathogen. Vaccines can take many forms. Vaccines can be whole organisms that share important antigens with the pathogen but are not pathogenic themselves (e.g., cowpox). Vaccines can also be prepared from killed (e.g., Salk polio vaccine) or attenuated (those that have lost the ability to produce disease, such as Sabin polio vaccine). Vaccines can also be prepared from purified macromolecules isolated from pathogenic organisms. For example, toxoid vaccines (e.g., tetanus and diphtheria) contain inactivated forms of soluble bacterial toxins that lead to the production of anti-toxin antibodies, but are not immune to the whole bacterium. Subunit vaccines (e.g., hepatitis B virus) contain only a single immunogenic protein isolated from the pathogen of interest. Hapten-conjugated vaccines attach certain carbohydrate or polypeptide epitopes isolated from the pathogen of interest to an immunogenic carrier, such as tetanus toxoid. These strategies essentially use the epitope as a hapten to induce antibody production, which then recognizes the same epitope of the natural pathogen. However, for maximal efficacy, such vaccines must contain both B and T cell epitopes, and the T cell epitopes must be selected to ensure that they can be recognized, presented, and responded to by the host individual's immune system.

DNA vaccines exploit the ability of host cells to take up and express DNA encoding disease-causing proteins injected intramuscularly.

Examples of antiviral vaccines that can be used in combination with the anti-PD-L1 antibodies used in the methods described herein include: Pevion Biotech's HCV vaccine (virasome), Transgene viron's TG4040 (MVA-HCV) designed to enhance cellular (cytotoxic T lymphocyte CD4+ and CD8+) immune responses against NS3, NS4 and NS5B, Inovio Biomedical's codon-optimized NS3/4a DNA vaccine, Novartis' HCV/CpG vaccine, GI-5005 - Globeimmune's HCV vaccine, IC41, Intercell's mixture of HCV CD4 and CD8 T epitopes combined with poly-L-arginine.

If administered as a mixture with an adjuvant, the host response to the immunogen can be enhanced. Immunological adjuvants function in one or more of the following ways: (1) prolonging the retention of the immunogen, (2) increasing the effective size of the immunogen (thereby promoting phagocytosis and presentation to macrophages), (3) stimulating the influx of macrophages or other immune cells to the injection site, or (4) promoting local cytokine production and other immunological activities. Examples of adjuvants include complete Freund's adjuvant (CFA), aluminum salts, and mycobacterium-derived proteins such as muramyl dipeptides or tripeptides.

The term "antibody" includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with multiple epitope specificities, multispecific antibodies (e.g., bispecific antibodies), diabodies and single-chain molecules, as well as antibody fragments (e.g., Fab, F(ab') ₂ , and Fv). The terms "immunoglobulin" (Ig) and "antibody" are used interchangeably herein.

The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies are composed of five basic heterotetrameric units and an additional polypeptide called a J chain, containing 10 antigen-binding sites; whereas IgA antibodies contain 2-5 basic four-chain units, which can be polymerized in combination with J chains to form multivalent assemblies. In the case of IgG, the four-chain unit is typically approximately 150,000 daltons. Each light chain is linked to a heavy chain by a single covalent disulfide bond, while the two heavy chains are linked to each other by one or more disulfide bonds, the number of which depends on the heavy chain isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain ( _VH ) at the N-terminus, followed by three (for each of the α and γ chains) and four (for the μ and ε isotypes) constant domains ( _CH ). Each light chain has a variable domain ( _VL ) at its N-terminus, followed by a constant domain at its other end. _{The VL} is aligned with the _VH , while the _CL is aligned with the first constant domain ( _CH1 ) of the heavy chain. Specific amino acid residues are believed to form an interface between the light and heavy chain variable domains. The paired _VH and _VL together form an antigen-binding site. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology , 8th edition, Daniel P. Sties, Abba I. Terr, and Tristram G. Parsolw (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6. Light chains from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Immunoglobulins can be assigned to different classes, or isotypes, based on the amino acid sequence of their heavy chain constant domain ( _CH ). There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses based on relatively minor differences in C _H sequence and function; for example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

An "isolated" antibody is one that has been identified, separated and/or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, an isolated polypeptide is free from all other components of its production environment. Contaminating components of its production environment (such as from recombinant transfected cells) are substances that will typically interfere with research, diagnostic or therapeutic applications of the antibody and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified: (1) to greater than 95% by weight of the antibody as measured by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator, or (3) to homogeneity as determined by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue (or preferably silver stain). Isolated antibodies include antibodies in situ within recombinant cells because at least one component of the antibody's natural environment will not be present. However, isolated polypeptides or antibodies are typically prepared by at least one purification step.

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody. The variable domains of the heavy and light chains may be referred to as "VH" and "VL," respectively. These domains are generally the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen-binding site.

The term "variable" refers to the situation where certain segments in the variable domain vary widely in antibody sequence. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not evenly distributed across all amino acids spanning the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) (found in both the light and heavy chain variable domains). The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of native heavy and light chains each contain four FR regions, which mostly adopt a β-sheet conformation and are connected by three HVRs that form loops connecting and, in some cases, forming part of the β-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, together with the HVRs of the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, Fifth Edition. National Institute of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cell-mediated cytotoxicity.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a group of substantially homogeneous antibodies, i.e., the individual antibodies constituting the group are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerizations, amidations) that may exist in small amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Compared to polyclonal antibody preparations (which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Except for their specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous antibody group and should not be construed as requiring the production of antibodies by any ad hoc method. For example, the monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Biology (2001); Hybridomas (monoclonal antibodies and T cell hybridomas) 563-681, (Elsevier, N.Y., 1981)), recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567), phage display technology (see, for example, Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol., 222: 581-597 (1992); Sidhu et al., J. Mol. Biol., 338(2): 299-310 (200 4); Lee et al., J. Mol. Biol. (Journal of Molecular Biology) 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA (Proceedings of the National Academy of Sciences of the United States of America) 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods (Journal of Immunological Methods) 284(1-2):119-132 (2004), and techniques for generating human or human-like antibodies from animals having part or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA (Proceedings of the National Academy of Sciences of the United States of America), 90:2551 (1993); Jakobovits et al., Nature (Nature), 362:255-258 (1993); Bruggemann et al., Year in Immunol., 7:33 (1993); U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016, Marks et al., Bio/Technology, 10:779-783 (1992); Lonberg et al., Nature, 368:856-859 (1994); Morrison, Nature, 368:812-813 (1994); Fishwild et al., Nature Biotechnol., 14:845-851 (1996); Neuberger, Nature Biotechnol., 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol., 13:65-93 (1995).

The term "naked antibody" refers to an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

The terms "full-length antibody," "intact antibody" or "complete antibody" are used interchangeably and refer to an antibody in its substantially intact form (as opposed to an antibody fragment). Specifically, complete antibodies include those having heavy and light chains including an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, an intact antibody may have one or more effector functions.

"Antibody fragments" comprise a portion of an intact antibody, preferably the antigen-binding and/or variable regions of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , and Fv fragments; diabodies; linear antibodies (see U.S. Patent 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments, and a residual "Fc" fragment, the name reflecting its ability to crystallize readily. The Fab fragment consists of an intact light chain and heavy chain variable domain ( _VH ) and one heavy chain first constant domain ( _CH1 ). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody produces a larger F(ab') ₂ fragment, which is roughly equivalent to two Fab fragments linked by disulfide bonds, having different antigen-binding activities and still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by the addition of additional residues at the carboxyl terminus of the _CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue of the constant domains bears a free thiol group. F(ab') ₂ antibody fragments originally were produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The Fc fragment comprises the carboxyl-terminal portions of two heavy chains held together by disulfide bonds.The effector functions of antibodies are determined by sequences in the Fc region, which is also the region recognized by Fc receptors (FcR) found on certain cell types.

"Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. From the folding of these two domains protrude six hypervariable loops (three loops each from the heavy and light chains) that contribute the amino acid residues that bind to the antigen and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv containing only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site.

"Single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising the _VH and _VL domains of an antibody linked into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the _VH and _VL domains to enable the sFv to form the desired antigen-binding structure. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

The "functional fragment" of the antibody of the present invention includes a portion of an intact antibody, generally including the antigen binding or variable region of the intact antibody, or the Fc region of an antibody, which retains or has modified FcR binding ability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

The term "diabodies" refers to small antibody fragments prepared by constructing sFv fragments (see previous paragraph) between the _VH and _VL domains using short linkers (approximately 5-10 residues) to achieve interchain, rather than intrachain, pairing of the V domains, thereby generating bivalent fragments, i.e., fragments with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossed" sFv fragments, in which the _VH and _VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described in more detail in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA (Proceedings of the National Academy of Sciences of the United States of America), 90: 6444-6448 (1993).

Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Nat. Acad. Sci. USA (Proceedings of the National Academy of Sciences of the United States of America) 81: 6851-6855 (1984)). Chimeric antibodies of interest herein include antibodies in which the antigen-binding region of the antibody is derived from an antibody produced by, for example, immunizing a macaque with an antigen of interest. As used herein, "humanized antibody" is used as a subset of "chimeric antibody".

"Humanized" forms of non-human (e.g., mouse) antibodies refer to chimeric antibodies that minimally contain sequences derived from non-human immunoglobulins. In one embodiment, a humanized antibody is a human immunoglobulin (receptor antibody) in which the HVR residues (hereafter defined) of the receptor are replaced with HVR residues of a non-human species (donor antibody) (such as mouse, rat, rabbit, or non-human primate) with desired specificity, affinity, and/or ability. In some cases, the framework (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, the humanized antibody may include residues that are not found in the receptor antibody or in the donor antibody. These modifications may be made in order to further improve the performance of the antibody, such as binding affinity. In general, a humanized antibody will comprise substantially all of at least one, typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more single FR residue substitutions that improve antibody performance (e.g., binding affinity, isomerization, immunogenicity, etc.). The number of such amino acid substitutions in the FRs generally does not exceed six in the H chain and three in the L chain. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, eg, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1: 105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5: 428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

"Human antibody" refers to an antibody that has an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or is produced using any of the techniques disclosed herein for producing human antibodies. This definition of a human antibody specifically excludes humanized antibodies that comprise non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol. 227: 381 (1991); Marks et al., J. Mol. Biol. 222: 581 (1991). Available methods for preparing human monoclonal antibodies are described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol. 147 (1): 86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, such as immunized xenograft mice (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 for XENOMOUSE ^™ technology). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), for human antibodies produced by human B cell hybridoma technology.

The terms "hypervariable region,""HVR," or "HV," as used herein, refer to regions of an antibody variable domain that are highly variable in sequence and/or form structurally defined loops. Typically, antibodies comprise six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In natural antibodies, H3 and L3 display the greatest diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Xu et al. Immunity 13:37-45 (2000); Johnson and Wu in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camelid antibodies composed only of heavy chains are functional and stable in the absence of light chains. See, eg, Hamers-Casterman et al. Nature 363 :446-448 (1993); Sheriff et al. Nature Struct. Biol. 3 :733-736 (1996).

[0015] A number of HVR descriptions are used and encompassed herein. The Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia instead refers to the positions of structural loops (Chothia and Lesk, J. Mol. Biol. 196 :901-917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. The "contact" HVRs are based on analysis of available complex crystal structures. The residues in each of these HVRs are documented below.

HVRs may include "extended HVRs" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. For each of these definitions, the variable domain residues are numbered according to Kabat et al., supra.

The term "variable domain residue numbering according to Kabat" or "amino acid position numbering according to Kabat" and variations thereof refer to the numbering system used by Kabat et al., supra, for heavy chain variable domain or light chain variable domain compilation of antibody sequences. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening or insertion of a variable domain FR or HVR. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat residue numbering of a given antibody can be determined by aligning the antibody sequence with the "standard" Kabat numbering sequence for regions of homology.

"Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

"Human consensus framework" or "acceptor human framework" refers to a framework that represents the most frequently occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of a human immunoglobulin VL or VH sequence is from a subtype of variable domain sequences. Generally, the subtype of the sequence is a subtype as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include, for VL, the subtype may be subtype kappa I, kappa II, kappa III, or kappa IV as described in Kabat et al. (supra). Additionally, for VH, the subtype may be subtype I, subtype II, or subtype III as described in Kabat et al. (supra). Alternatively, a human consensus framework can be obtained as described above, wherein specific residues, such as human framework residues, are selected based on their homology to a donor framework by aligning the donor framework with a collection of multiple human framework sequences. An acceptor human framework "derived from" a human immunoglobulin framework or human consensus framework can comprise the same amino acid sequence thereof, or it can comprise changes in a pre-existing amino acid sequence. In some embodiments, the number of pre-existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

A "VH subgroup III consensus framework" comprises a consensus sequence obtained from the amino acid sequences in variable heavy chain subgroup III of Kabat et al. (supra). In one embodiment, the VH subgroup III consensus framework amino acid sequence comprises at least a portion or all of each of the following sequences: EVQLVESGGGLVQPGGSLRLSCAAS (HC-FR1) (SEQ ID NO:4), WVRQAPGKGLEWV (HC-FR2), (SEQ ID NO:5), RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (HC-FR3, SEQ ID NO:6), WGQGTLVTVSA (HC-FR4), (SEQ ID NO:7).

A "VLκI consensus framework" comprises a consensus sequence obtained from the amino acid sequence of variable light chain kappa subgroup I in Kabat et al. (supra). In one embodiment, the VH subgroup I consensus framework amino acid sequence comprises at least a portion or all of each of the following sequences: DIQMTQSPSSLSASVGDRVTITC (LC-FR1) (SEQ ID NO: 11), WYQQKPGKAPKLLIY (LC-FR2) (SEQ ID NO: 12), GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (LC-FR3) (SEQ ID NO: 13), FGQGTKVEIKR (LC-FR4) (SEQ ID NO: 14).

"Amino acid modification" at a designated site (e.g., in the Fc region) refers to the replacement or deletion of a designated residue, or the insertion of at least one amino acid residue near a designated residue. Insertion "near" a designated residue means insertion within one to two residues thereof. Insertion can be at the N-terminus or C-terminus of a designated residue. Preferred amino acid modifications herein are replacements.

An "affinity matured" antibody is one that has one or more alterations in one or more HVRs that result in an increase in the affinity of the antibody for the antigen compared to a parent antibody that does not possess these alterations. In one embodiment, affinity matured antibodies have nanomolar or even picomolar avidity for the target antigen. Affinity matured antibodies can be generated by methods known in the art. For example, Marks et al., Bio/Technology 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of HVR and/or framework residues is described in, for example, Barbas et al., Proc. Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

As used herein, the term "specific binding" or "specific for" refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of the target in the presence of a population of heterologous molecules (including biomolecules). For example, an antibody that specifically binds to a target (which may be an epitope) is an antibody that binds to the target with greater affinity, mobility, more readily, and/or for a longer period of time than it binds to other targets. In one embodiment, the degree of binding of the antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In certain embodiments, the antibody specifically binds to an epitope on a protein that is conserved between proteins from different species. In another embodiment, specific binding may include, but is not necessarily, exclusive binding.

A "blocking antibody" or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. In some embodiments, a blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen. The anti-PD-L1 antibodies of the present invention block signaling through PD-1, thereby restoring the functional response of T cells to antigen stimulation from a dysfunctional state.

An "agonistic" or activating antibody is one that enhances or initiates signaling of an antigen to which it binds. In some embodiments, an agonistic antibody causes or activates signaling in the absence of a natural ligand.

The term "solid phase" describes a non-aqueous matrix to which the antibodies of the present invention can be attached. Examples of solid phases encompassed herein include those made partially or completely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamide, polystyrene, polyvinyl alcohol, and silicone. In certain embodiments, depending on the context, the solid phase may comprise the wells of an assay plate; in other embodiments, it refers to a purification column (e.g., an affinity chromatography column). The term also includes discontinuous solid phases of discrete particles, such as those described in U.S. Patent No. 4,275,149.

"Antibody effector function" refers to those biological activities attributable to the Fc region of an antibody (a native sequence Fc region or an amino acid sequence variant Fc region), and varies with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. "Reducing or minimizing" an antibody effector function means that it is reduced by at least 50% (alternatively 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) from that of a wild-type or unmodified antibody. One of ordinary skill in the art can readily determine and measure the effector functions of an antibody. In a preferred embodiment, the effector functions of complement fixation, complement-dependent cytotoxicity, and antibody-dependent cellular toxicity of the antibody are affected. In some embodiments of the present invention, the elimination of effector function is accomplished by a mutation in the constant region that eliminates glycosylation (e.g., a "poor effector mutation"). In one aspect, the poor effector mutation is an N297A or DANA mutation (D265A+N297A) in the CH2 region. Shields et al., J. Biol. Chem. 276 (9): 6591-6604 (2001). Alternatively, other mutations that result in a reduction or elimination of effector function include: K322A and L234A/L235A (LALA). Alternatively, the reduction or elimination of effector function can be accomplished by production techniques, such as expression in a non-glycosylated host cell (e.g., E. coli), or in a host cell that results in an altered pattern of ineffective or inefficient promotion of effector function (e.g., Shinkawa et al., J. Biol. Chem. 278 (5): 3466-3473 (2003).

"Antibody-dependent cell-mediated cytotoxicity" or ADCC refers to a form of cytotoxicity in which secreted Ig bound by Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells, which are then killed with cytotoxins. The antibodies "arm" the cytotoxic cells and are necessary for killing the target cells by this mechanism. The primary cells mediating ADCC (NK cells) express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Fc expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9 :457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337, may be performed. Effector cells that can be used for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the target molecule can be assessed in vivo, for example, in an animal model, such as that disclosed by Clynes et al. in PNAS USA 95 : 652-656 (1998).

Unless otherwise indicated herein, the residue numbering of immunoglobulin heavy chains is that of the EU index as in Kabat et al., supra. "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the immunoglobulin heavy chain Fc region can vary, the human IgG heavy chain Fc region is generally defined as the segment from its amino acid residue at Cys226 or Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during the production or purification of the antibody, or by recombinantly modifying the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies can include antibody groups in which all K447 residues have been removed, antibody groups in which no K447 residue has been removed, and antibody groups having a mixture of antibodies with and without K447 residues. Suitable native sequence Fc regions for use in the antibodies of the present invention include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. In addition, a preferred FcR is an FcR (gamma receptor) that binds to an IgG antibody, and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see M. Annu. Rev. Immunol. 15 :203-234 (1997). For review of FcRs, see Ravetch and Kinet, Annu. Rev. Immunol. 9 :457-92 (1991); Capel et al., Immunomethods 4 :25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126 :330-41 (1995). The term "FcR" herein encompasses additional FcRs, including those to be identified in the future.

The term "Fc receptor" or "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus. Guyer et al., J. Immunol. 117 :587 (1976) and Kim et al., J. Immunol. 24 :249 (1994). Methods for measuring binding to FcRn are known (see, for example, Ghetie and Ward., Immunol. Today 18 (12):592-8 (1997); Ghetie et al., Nature Biotechnology, 15 (7):637-40 (1997); Hinton et al., J. Biol. Chem. 279 (8):6213-6 (2004); WO 2004/92219 (Hinton et al.)). The in vivo binding and serum half-life of high-affinity human FcRn binding polypeptides can be determined, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered polypeptides with variant Fc regions. WO 2004/42072 (Presta) describes antibody variants with improved or reduced FcR binding. See also, for example, Shields et al., J. Biol. Chem. 9 (2):6591-6604 (2001).

"Effecter cells" refer to leukocytes that express one or more FcRs and perform effector functions. On the one hand, effector cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources, such as blood. Effector cells are typically lymphocytes associated with the effector phase, and function to produce cytokines (helper T cells), kill pathogen-infected cells (cytotoxic T cells), or secrete antibodies (differentiated B cells).

"Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass) bound to their cognate antigen. To assess complement activation, a CDC assay, such as that described by Gazzano-Santoro et al., J. Immunol. Methods 202 :163 (1996), can be performed. Antibody variants with altered Fc region amino acid sequences and increased or decreased Clq binding are described in U.S. Patent No. 6,194,551 Bl and WO99/51642. The contents of those patent publications are expressly incorporated herein by reference. See also Idusogie et al., J. Immunol. 164:4178-4184 (2000).

The N-glycosylation site in IgG is Asn297 in the CH2 domain. The present invention also provides compositions of antigen-binding, humanized antibodies having an Fc region with reduced or no effector function. One way to accomplish this is the A297N substitution, which has previously been shown to eliminate complement binding and effector function in anti-CD20 antibodies ("poor effector Fc mutation"). Idusgie et al., supra. Due to this mutation, anti-PD-L1 antibodies of the present invention containing this Fc mutation produced in mammalian cells such as CHO will not have any glycosylation, which in turn leads to reduced or minimal effector function. Alternatively, antibody effector function can be eliminated by expression in non-mammalian cells such as E. coli without the CH2 substitution.

"Binding affinity" generally refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of a dissociation constant (Kd). Avidity can be measured by conventional methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigen more rapidly and tend to remain bound for longer periods of time. A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention. Specific instructions and exemplary embodiments for measuring binding affinity are described below.

In one embodiment, the "Kd" or "Kd value" according to the present invention is measured by a radiolabeled antigen binding assay (RIA) performed with a Fab form of an antibody and an antigen molecule as described in the following assay: The assay measures the solution binding affinity of Fabs for antigen by equilibrating the Fab with a minimal concentration of ( ¹²⁵ I)-labeled antigen in the presence of a titration series of unlabeled antigen, followed by capturing the bound antigen with an anti-Fab antibody-coated plate (Chen, et al., (1999) J. Mol. Biol. 293: 865-881). To determine the conditions of the assay, microtiter plates (Dynex) were coated overnight with 5 μg/ml of a capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and subsequently blocked with 2% (w/v) bovine serum albumin in PBS at room temperature (approximately 23° C.) for 2-5 hours. In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [ ¹²⁵ I]-antigen was mixed with a serial dilution of the Fab of interest (consistent with the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al. (1997) Cancer Res. 57:4593-4599). The Fab of interest was then incubated overnight; however, the incubation could be continued for a longer period (e.g., 65 hours) to ensure that equilibrium was reached. The mixture was then transferred to a capture plate for incubation at room temperature for 1 hour. The solution was then removed and the plate was washed 8 times with 0.1% Tween-20 in PBS. When the plate had dried, 150 μl/well of scintillant (MicroScint-20; Packard) was added and the plate was counted for 10 minutes in a Topcount gamma counter (Packard). Concentrations of each Fab that provided less than or equal to 20% of maximal binding were selected for use in the competitive binding assay.

According to another embodiment, Kd is measured by surface plasmon resonance assay using a BIAcore ® -2000 or -3000 instrument (BIAcore, Inc., Piscataway, NJ) at 25°C using an immobilized antigen CM5 chip at approximately 10 response units (RU). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIAcore Inc.) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxy-succinimide (NHS) according to the supplier's instructions. Antigen was diluted to 5 μg/ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl/min to obtain approximately 10 response units (RU) of coupled protein. Following antigen injection, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS containing 0.05% TWEEN 20 ^™ surfactant (PBST) were injected at 25° C. at a flow rate of approximately 25 μl/min. Association rates (k _on ) and dissociation rates (k _off ) were calculated using a simple one-to-one Langmuir binding model (Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) was calculated as the ratio k _off /k _on . See, e.g., Chen et al., J. Mol. Biol. 293: 865-881 (1999). If the on-rate exceeds 10 ⁶ M ^-1 s ^-1 according to the surface plasmon resonance assay above, the on-rate can be determined using a fluorescence quenching technique, i.e., measuring the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen ^antibody (Fab form) in PBS, pH 7.2 at 25°C in the presence of increasing concentrations of antigen as measured in a spectrometer such as a spectrophotometer equipped with a cut-off device (Aviv Instruments) or a 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) using a stirred cuvette.

The "on-rate" (rate of association) or "k _on " according to the present invention can also be determined as described above using a BIAcore-2000 or -3000 system (BIAcore, Inc., Piscataway, NJ) at 25°C using an immobilized antigen CM5 chip at approximately 10 response units (RU). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIAcore Inc.) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (ECD) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen was diluted to 5 mg/ml (approximately 0.2 mM) with 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 ml/min to obtain approximately 10 response units (RU) of coupled protein. After antigen injection, 1 M ethanolamine was added to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS containing 0.05% Tween 20 (PBST) were injected at a flow rate of approximately 25 ul/min at 25°C. Association rates (k _on ) and dissociation rates (k _off ) were calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2). The equilibrium dissociation constant (Kd) was calculated as the ratio k _off /k _on . See, e.g., Chen, Y. et al. (1999) J. Mol. Biol. 293 : 865-881. However, if the on-rate exceeds 10 ⁶ M ^-1 S ^-1 according to the surface plasmon resonance assay above, the on-rate is preferably determined using a fluorescence quenching technique, i.e., measuring the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 in the presence of increasing concentrations of antigen at 25°C as measured in a spectrometer such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000 series SLM-Aminco spectrophotometer (ThermoSpectronic) with a stirred cuvette.

The term "substantially reduced" or "substantially different," as used herein, refers to a sufficiently high degree of difference between two values (typically one associated with a molecule and the other associated with a reference/comparator molecule) such that one of skill in the art would consider the difference between the two values to be statistically significant in the context of the biological characteristic measured by the value (e.g., Kd value). The difference between the two values as a function of the value of the reference/comparator molecule is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50%.

The term "substantially similar" or "substantially identical," as used herein, refers to a sufficiently high degree of similarity between two values (e.g., one associated with an antibody of the invention and the other associated with a reference/comparator antibody) such that one of skill in the art would consider the difference between the two values to be minimal or biologically and/or statistically insignificant in the context of the biological characteristic measured by the value (e.g., Kd value). The difference between the two values as a function of the reference/comparator value is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and/or less than about 10%.

"Percent (%) amino acid sequence identity" and "homology" with respect to peptide, polypeptide or antibody sequences are defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specific peptide or polypeptide sequence, after aligning the candidate sequence with the specific peptide or polypeptide sequence (and introducing gaps, if necessary) to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignments for determining percent amino acid sequence identity can be performed using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN ^™ (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignments, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared. However, for this purpose, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, which was developed by Genentech, Inc. The source code for ALIGN-2 has been submitted with user documentation to the U.S. Copyright Office (Washington DC, 20559) under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc. (South San Francisco, California). The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. The ALIGN-2 program sets all sequence alignment parameters and does not change.

In the case where ALIGN-2 is applied to amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (or in other words: a given amino acid sequence A has or contains a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

Multiply the X/Y ratio by 100

where X is the number of amino acid residues scored as identical matches using the sequence alignment program ALIGN-2 in an alignment of A and B in that program, and where Y is the total number of amino acid residues in B. It will be understood that when amino acid sequence A is not of equal length to amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A.

Unless specifically stated otherwise, all % amino acid sequence identity values used herein are derived using the ALIGN-2 computer program as described in the preceding paragraph.

An "isolated" nucleic acid molecule encoding an antibody herein is one that has been identified and separated from at least one contaminating nucleic acid molecule with which it is normally associated in the environment in which it was produced. Preferably, the isolated nucleic acid is free from all components associated with the environment in which it was produced. An isolated nucleic acid molecule encoding the polypeptides and antibodies herein is in a form that is different from the form or arrangement in which it is found in nature. Thus, an isolated nucleic acid molecule is distinguished from nucleic acids encoding the polypeptides and antibodies herein that are naturally present in cells.

The term "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. For example, control sequences suitable for prokaryotes include a promoter, an optional operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

A nucleic acid is "operably linked" if it is placed in a functional relationship with another nucleic acid sequence. For example, a presequence or secretory leader DNA is operably linked to a polypeptide DNA if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to promote translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in the same reading frame. However, enhancers do not have to be contiguous. Linking can be achieved by ligation at convenient restriction sites. If such sites are not available, synthetic oligonucleotide adapters or linkers can be used in accordance with conventional practice.

The term "epitope-tagged" as used herein refers to a chimeric polypeptide comprising a polypeptide or antibody described herein fused to a "tag polypeptide". The tag polypeptide has enough residues to provide an epitope against which an antibody can be prepared, but is short enough so that it does not interfere with the activity of the polypeptide to which it is fused. The tag polypeptide is preferably also quite unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides typically have at least 6 amino acid residues and typically between about 8 and about 50 amino acid residues (preferably between about 10 and 20 amino acid residues).

As used herein, the term "immunoadhesin" refers to an antibody-like molecule that combines the binding specificity of a heterologous protein ("adhesin") with the effector function of an immunoglobulin constant domain. Structurally, an immunoadhesin comprises a fusion of an amino acid sequence with a desired binding specificity that is different from the antigen recognition and binding site of an antibody (i.e., "heterologous") and an immunoglobulin constant domain sequence. The adhesin portion of an immunoadhesin molecule is typically a continuous amino acid sequence that comprises at least the binding site for a receptor or ligand. The immunoglobulin constant domain sequence in an immunoadhesin can be obtained from any immunoglobulin, such as IgG-1, IgG-2 (including IgG2A and IgG2B), IgG-3 or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM. Ig fusions preferably comprise replacement of at least one variable region within an Ig molecule with a domain of a polypeptide or antibody as described herein. In a particularly preferred embodiment, the immunoglobulin fusion comprises the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgG1 molecule. See also U.S. Patent No. 5,428,130, issued June 27, 1995, for the production of immunoglobulin fusions. For example, useful immunoadhesins as second agents for use in combination therapies herein include polypeptides comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 (or vice versa) fused to a constant domain of an immunoglobulin sequence.

A "fusion protein" or "fusion polypeptide" refers to a polypeptide having two parts covalently linked together, wherein each part is a polypeptide with a different property. The property can be a biological property, such as in vitro or in vivo activity. The property can also be a simple chemical or physical property, such as binding to a target molecule, catalysis of a reaction, etc. The two parts can be directly linked to each other in reading frame by a single peptide bond or by a peptide linker.

A "stable" formulation is one in which the protein substantially maintains its physical and chemical stability and integrity during storage. Various analytical techniques for measuring protein stability are available in the art, as reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee, ed., Marcel Dekker, Inc., New York, New York, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10 : 29-90 (1993). Stability can be measured at a selected temperature for a selected period of time. For rapid screening, the formulation can be maintained at 40°C for 2 weeks to 1 month, during which time stability is measured. When the formulation is to be stored at 2-8°C, the formulation should generally be stable at 30°C or 40°C for at least 1 month and/or at 2-8°C for at least 2 years. When the formulation is to be stored at 30°C, the formulation should generally be stable at 30°C for at least 2 years and/or at 40°C for at least 6 months. For example, the degree of aggregation during storage can be used as an indicator of protein stability. Thus, a "stable" formulation can be one in which less than about 10%, and preferably less than about 5%, of the protein in the formulation is present as aggregates. In other embodiments, any increase in aggregate formation during storage of the formulation can be determined.

A "reconstituted" formulation is one that has been prepared by dissolving a lyophilized protein or antibody formulation in a diluent to disperse the protein throughout the formulation. The reconstituted formulation is suitable for administration (e.g., subcutaneous administration) to a patient in need of treatment with the protein of interest and, in certain embodiments of the invention, may be a formulation suitable for parenteral or intravenous administration.

An "isotonic" formulation is one that has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of from about 250 to 350 mOsm. The term "hypotonic" describes a formulation that has an osmotic pressure lower than that of human blood. Correspondingly, the term "hypertonic" is used to describe a formulation that has an osmotic pressure higher than that of human blood. For example, isotonicity can be measured using a vapor pressure or ice-type osmometer. The formulations of the present invention are hypertonic as a result of the addition of salts and/or buffers.

As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cells or mammals to which they are exposed at the dosages and concentrations employed. Typically, a physiologically acceptable carrier is a pH-buffered aqueous solution. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN ^™ , polyethylene glycol (PEG), and PLURONICS ^™ .

"Package insert" means an instruction sheet customarily included in commercial packages of drugs, which contains information about the indications, usage, dosage, administration, contraindications, other drugs to be combined with the packaged drug and/or warnings concerning the use of such drugs.

"Pharmaceutically acceptable acids" include inorganic and organic acids that are non-toxic at the concentrations and in the manner in which they are formulated. For example, suitable inorganic acids include hydrochloric acid, perchloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, sulfonic acid, sulfinic acid, sulfanilic acid, phosphoric acid, carbonic acid, and the like. Suitable organic acids include straight-chain and branched hydrocarbon, aromatic, cyclic, cycloaliphatic, araliphatic, heterocyclic, saturated, unsaturated, monocarboxylic, dicarboxylic, and tricarboxylic acids, including, for example, formic acid, acetic acid, 2-hydroxyacetic acid, trifluoroacetic acid, phenylacetic acid, trimethylacetic acid, t-butylacetic acid, anthranilic acid, propionic acid, 2-hydroxypropionic acid, 2-oxopropionic acid, malonic acid, cyclopentanepropionic ... propionic), 3-phenylpropionic acid, butyric acid, succinic acid, benzoic acid, 3-(4-hydroxybenzyl)benzoic acid, 2-acetoxybenzoic acid, ascorbic acid, cinnamic acid, dodecylsulfuric acid, stearic acid, muconic acid, mandelic acid, succinic acid, pamoic acid, fumaric acid, malic acid, maleic acid, hydroxymaleic acid, malonic acid, lactic acid, citric acid, tartaric acid, glycolic acid, aldonic acid, gluconic acid, pyruvic acid, glyoxylic acid, oxalic acid, methanesulfonic acid, succinic acid, salicylic acid, phthalic acid, palmitic acid, palmeic acid, thiocyanic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid (4-chorobenzenesulfonic acid) acid), naphthalene-2-sulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo(2.2.2)-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4'-methylenebis-3-(hydroxy-2-ene-1-carboxylic acid), hydroxynaphthoic acid.

"Pharmaceutically acceptable bases" include inorganic and organic bases that are non-toxic at the concentrations and manner in which they are formulated. For example, suitable bases include those formed from inorganic alkali metals, such as lithium, sodium, potassium, magnesium, calcium, ammonium, iron, zinc, copper, manganese, aluminum, N-methylglucamine, morpholine, piperidine, and organic non-toxic bases, including primary, secondary, and tertiary amines, substituted amines, cyclic amines, and basic ion exchange resins [e.g., ^N (R') ₄₊ (wherein R' is independently H or _C1-4 hydrocarbon group, e.g., ammonium, Tris)], for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, meglumine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Particularly preferred organic non-toxic bases are isopropylamine, diethylamine, ethanolamine, trimethamine, dicyclohexylamine, choline and caffeine. Additional pharmaceutically acceptable acids and bases useful in the present invention include those derived from amino acids, such as histidine, glycine, phenylalanine, aspartic acid, glutamic acid, lysine and asparagine.

"Pharmaceutically acceptable" buffers and salts include those derived from acid addition salts and base addition salts of the acids and bases indicated above. Specific buffers and/or salts include histidine, succinate, and acetate.

A "pharmaceutically acceptable sugar" is a molecule that, when combined with a protein of interest, effectively prevents or reduces chemical and/or physical instability of the protein during storage. A "pharmaceutically acceptable sugar" may also serve as a "lyoprotectant" when the formulation is to be lyophilized and then reconstituted. Exemplary sugars and their corresponding sugar alcohols include: amino acids, such as monosodium glutamate or histidine; methylamines, such as betaine; lyotropic salts, such as magnesium sulfate; polyols, such as tribasic or higher molecular weight sugar alcohols, such as glycerin, dextran, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol; propylene glycol; polyethylene glycol; and combinations thereof. Additional exemplary lyoprotectants include glycerol and gelatin, and melibiose, melezitose, raffinose, mannotriose, and stachyose. Examples of reducing sugars include glucose, maltose, lactose, maltulose, isomaltulose, and lactulose. Examples of non-reducing sugars include glycosides of non-reducing polyols selected from sugar alcohols and other linear polyols. Preferred sugar alcohols are monoglycosides, particularly those obtained by reducing disaccharides such as lactose, maltose, lactulose and maltulose. The side groups of the glycosides may be glucosidic or galactosidic. Other examples of sugar alcohols are glucitol, maltitol, lactitol and isomaltulose. Preferred pharmaceutically acceptable sugars are non-reducing sugars trehalose or sucrose. Adding a pharmaceutically acceptable sugar to the formulation (e.g., before lyophilization) in a "protective amount" means that the protein substantially maintains its physical and chemical stability and integrity during storage (e.g., after reconstitution and storage).

The "diluent" of interest herein is a pharmaceutically acceptable (safe and non-toxic for administration to humans) agent useful for preparing a liquid formulation, such as a formulation to be reconstituted after lyophilization. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution. In alternative embodiments, the diluent may include an aqueous solution of a salt and/or a buffer.

A "preservative" is a compound that can be added to the formulations herein to reduce bacterial activity. The addition of a preservative can, for example, assist in the production of a formulation for multiple uses (multiple doses). Examples of possible preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols, such as phenol, butyl and benzyl alcohols, alkyl parabens, such as methyl or propyl parabens, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The most preferred preservative herein is benzyl alcohol.

"Treatment" refers to clinical intervention designed to alter the natural course of the individual or cell to be treated, and can be performed for prevention or in the course of clinical pathology. Desirable effects of treatment include preventing the occurrence or recurrence of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and symptom relief or improved prognosis. In some embodiments, the antibodies of the present invention are used to delay the development of a disease or disorder. For example, if one or more symptoms associated with a T cell dysfunction disorder are alleviated using the apoptotic anti-PD-L1 antibodies of the present invention, the subject is successfully "treated."

"Effective amount" refers to an amount effective to achieve at least the desired or indicated effect (including therapeutic or preventive effect) at the desired dosage and for the desired period of time. For example, an effective amount of an anti-PD-L1 antibody of the present invention is the minimum concentration that results in at least inhibition of signaling from PD-L1, such inhibition being carried out by PD-1 on T cells or B7.1 on other APCs, or both.

A "therapeutically effective amount" is at least the minimum concentration required to produce a measurable improvement or prevention of a particular condition. The therapeutically effective amount herein may vary depending on factors such as the patient's disease state, age, sex, and weight, as well as the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody are outweighed by the therapeutically beneficial effects. For example, a therapeutically effective amount of an anti-PD-L1 antibody of the invention is at least the minimum concentration that results in inhibition of at least one symptom of a T cell dysfunction disorder.

A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic effect, at the desired dosage and for the desired period of time. For example, a prophylactically effective amount of an anti-PD-L1 antibody of the present invention is at least the minimum concentration that prevents or reduces the development of at least one symptom of a T cell dysfunction disorder.

"Chronic" administration refers to administration of one or more drugs in a continuous fashion, as opposed to a short-term fashion, to maintain the initial therapeutic effect (activity) for an extended period of time. "Intermittent" administration refers to treatment that is not continuous without interruption, but rather is cyclical in nature.

For therapeutic purposes, "mammal" refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals, such as dogs, horses, rabbits, cows, pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc. Preferably, mammal refers to a human.

The term "pharmaceutical formulation" refers to a preparation that is in a form that permits the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to a subject to which the formulation is administered. The formulation is sterile.

A "sterile" preparation is sterile, or free from all viable microorganisms and their spores.

The term "about" is used herein to refer to the normal error range for the respective numerical value that is readily known to those skilled in the art.

An "autoimmune disease" is a disease or condition that is caused by and directed against an individual's own tissues or organs, or a co-isolation or manifestation thereof, or a condition resulting therefrom. An autoimmune disease can be an organ-specific disease (i.e., the immune response is specifically directed against one organ system, such as the endocrine system, hematopoietic system, skin, cardiopulmonary system, gastrointestinal and hepatic system, renal system, thyroid, ear, neuromuscular system, central nervous system, etc.) or a systemic disease that can affect multiple organ systems (e.g., systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), polymyositis, etc.). Preferred such diseases include autoimmune rheumatologic disorders (such as, for example, RA, Sjögren's syndrome, scleroderma, lupus such as SLE and lupus nephritis, polymyositis-dermatomyositis, cryoglobulinemia, anti-phospholipid antibody syndrome, and psoriatic arthritis), autoimmune gastrointestinal and liver disorders (such as, for example, inflammatory bowel diseases (e.g., ulcerative colitis and Crohn's disease), autoimmune gastritis and pernicious anemia, autoimmune hepatitis, primary biliary cirrhosis, and biliary cirrhosis), primary sclerosing cholangitis, and celiac disease), vasculitis (such as, for example, ANCA-negative vasculitis and ANCA-associated vasculitis, including Churg-Strauss vasculitis, Wegener's granulomatosis, and microscopic polyangiitis), autoimmune neurological disorders (such as, for example, multiple sclerosis, opsoclonus myoclonus syndrome, myasthenia gravis, neuromyelitis optica, Parkinson's disease, Alzheimer's disease, disease and autoimmune polyneuropathies), renal disorders (such as, for example, glomerulonephritis, Goodpasture's syndrome, and Berger's disease), autoimmune dermatologic disorders (such as, for example, psoriasis, urticaria, hives, pemphigus vulgaris, bullous pemphigoid, and cutaneous lupus erythematosus), hematologic disorders (such as, for example, thrombocytopenic purpura, thrombotic thrombocytopenic purpura, post-transfusion purpura), and autoimmune thyroid diseases (e.g., Graves' disease and thyroiditis). More preferred such diseases include, for example, RA, ulcerative colitis, ANCA-associated vasculitis, and autoimmune endocrine disorders. vasculitis), lupus, multiple sclerosis, Sjögren's syndrome, Graves' disease, IDDM, pernicious anemia, thyroiditis, and glomerulonephritis.

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and/or causes cell destruction. The term includes radioisotopes (e.g., At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² and radioisotopes of Lu) and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.

A "chemotherapeutic agent" is a compound that is effective for treating cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamin, and dapoxetine; e), trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol); beta-lapachone; lapachol; colchicines; betulinic acid acid; camptothecin (including the synthetic analogs topotecan CPT-11 (irinotecan), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; pemetrexed; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllinic acid acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189, and CB1-TM1); eleutherobin; pancratistatin; TLK-286; CDP323 (an oral alpha-4 integrin inhibitor); sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, and chlorambucil. hydrochloride), melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma and calicheamicin omega (see, e.g., Nicolaou et al., Angew, Chem. Int. .Ed.Engl.33:183-186 (1994)); enediyne anthracyclines, including enediyne anthracycline A; esperamicin; and neocarzinostatin chromophores and related pigment proteins (enediyne antibiotic chromophores), aclacinomycins, actinomycins, anthramycins, azaserine, bleomycins, cactinomycin C ( nomycin), carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin), ), cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposome doxorubicin hydrochloride and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine, tegafur, capecitabine, epothilone, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate, and oxazolidinone; etrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, and imatinib (a 2-phenylaminopyrimidine derivative), as well as other c-Kit inhibitors; anti-adrenals such as aminoglutethimide, mitotane, and trilostane; and folic acid supplements such as folinic acid. acid; aceglatone; aldophosphamide glycoside; 5-aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; polysaccharide complex (JHS Natural Products (JHS Natural Products), Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verracurin A, roridin A, and roridin B); A) and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");thiotepa; taxoids, such as paclitaxel; albumin-engineered nanoparticle formulations of paclitaxel (ABRAXANE ^™ ) and docetaxel); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine oxaliplatin; leucovovin; vinorelbine novantrone; edatrexate; daunomycin; aminopterin; ibandronate; and the topoisomerase inhibitor RFS. 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing; and combinations of two or more thereof, such as CHOP, which stands for a combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, which stands for a treatment regimen using oxaliplatin (ELOXATIN ^™ ) in combination with 5-FU and leucovovin. A particularly preferred chemotherapeutic agent for use in combination with the anti-PD-L1 antibodies of the present invention, particularly in the treatment of tumor immunotherapy, is gemcitabine.

Also included in this definition are anti-hormonal agents that act to regulate, reduce, block or inhibit the effects of hormones that can promote cancer growth, typically in the form of systemic or systemic therapy. They can be hormones themselves. Examples include antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene; anti-progesterones; estrogen receptor downregulators (ERDs); estrogen receptor antagonists such as fulvestrant; agents that act to suppress or shut down the ovaries, for example, luteinizing hormone-releasing hormone (LHRH) agonists such as leuprolide acetate (and leuprolide), goserelin acetate, buserelin acetate, and valproate. acetate) and tripterelin; antiandrogens such as flutamide, nilutamide, and bicalutamide; and aromatase inhibitors, which inhibit aromatase, the enzyme that regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestanie, fadrozole, vorozole, letrozole, and anastrozole. Additionally, the definition of chemotherapeutic agents includes bisphosphonates such as clodronate (e.g., clodronate or dapoxetine), etidronate NE-58095, zoledronic acid, and dapoxetine. acid)/zoledronate, alendronate, pamidronate, tiludronate, or risedronate, and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as, for example, PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as vaccines and gene therapy vaccines, For example, vaccines, vaccines and vaccines; topoisomerase 1 inhibitors (for example); anti-estrogens such as fulvestrant; Kit inhibitors such as imatinib or EXEL-0862 (tyrosine kinase inhibitors); EGFR inhibitors such as erlotinib or cetuximab; anti-VEGF inhibitors such as bevacizumab; arinotecan; rmRH (e.g., lapatinib and lapatinib ditosylate (a dual tyrosine kinase small molecule inhibitor of ErbB-2 and EGFR, also known as GW572016); 17AAG (a geldanamycin derivative, which is a heat shock protein (Hsp) 90 toxin), and pharmaceutically acceptable salts, acids or derivatives of any of the above substances.

"Growth inhibitor" refers to a compound or composition that inhibits cell growth (which growth depends on receptor activation in vitro or in vivo). Therefore, growth inhibitors include growth inhibitors that significantly reduce the percentage of receptor-dependent cells in the S phase. Examples of growth inhibitors include agents that block cell cycle progression (at stages other than the S phase), such as drugs that induce G1 arrest and M phase arrest. Classical M phase blockers include vincas and vinca alkaloids (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Agents that cause G1 arrest also cause S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in Murakami et al., The Molecular Basis of Cancer , Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" (WB Saunders: Philadelphia, 1995), particularly page 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs derived from the yew tree. Docetaxel (Rhone-Poulenc Rorer), derived from the European yew tree, is a semisynthetic analog of paclitaxel (Bristol-Myers Squibb).

The term "cytokine" is a general term that refers to proteins released by one cell population that act as intercellular regulators on another cell population. Examples of such cytokines are lymphokines, monokines; interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15, IL-35, including rIL-2; tumor necrosis factors such as TNF-α or TNF-β; and other polypeptide factors including LIF and kit ligand (KL), with the term "interleukin" now essentially becoming synonymous with cytokine. As used herein, the term cytokine includes proteins derived from natural sources or from recombinant cell culture, as well as biologically active equivalents of native sequence cytokines, including synthetically produced small molecule entities and pharmaceutically acceptable derivatives and salts thereof. Cytokines can be classified according to the proximity of their target of interest, where autocrine refers to acting on the same cells that secrete it, paracrine refers to the effect being limited to the immediate vicinity of the cytokine secreted, and endocrine refers to acting on the distal regions of the body. Immune cytokines can also be classified according to whether they enhance type I responses (e.g., IFN-γ, TGF-β, etc.), which promote cellular immunity, or enhance type II responses (IL-4, IL-10, IL-13, etc.), which promote antibodies or humoral immunity. Immune cytokines play a role in costimulation, maturation, proliferation, activation, inflammation, growth, differentiation, cytokine production and secretion, and the survival of a variety of immune cells.

The term "hormone" refers to a polypeptide hormone that is typically secreted by a glandular organ having a duct. Hormones include, for example, growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; estradiol; hormone replacement therapy; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, or testolactone; prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); prolactin, placental lactogen, mouse gonadotropin-related peptide, gonadotropin-releasing hormone; inhibins; activins; mullerian-inhibiting substance; and thrombopoietin. As used herein, the term hormone includes proteins from natural sources or from recombinant cell culture as well as biologically active equivalents of the native sequence hormone, including synthetically produced small molecule entities and pharmaceutically acceptable derivatives and salts thereof.

III. Modes for carrying out the invention

A. Humanization using phage display

Hypervariable region-grafted variants described herein are generated by Kunkel mutagenesis of nucleic acids encoding human acceptor sequences using separate oligonucleotides for each hypervariable region. Kunkel et al., Methods Enzymol. 154 : 367-382 (1987). Conventional techniques can be used to introduce appropriate changes into the framework and/or hypervariable regions to correct and reestablish appropriate hypervariable region-antigen interactions.

Phage (mid) display (also referred to herein as phage display) can be used as a convenient and rapid method for producing and screening many different possible variant antibodies in the library produced by sequence randomization. However, other methods for preparing and screening the antibodies that change are also available to the skilled person.

Phage (mid) display (also referred to herein as phage display in some environments) can be used as a convenient and rapid method for producing and screening many different possible variant antibodies in the library produced by sequence randomization. However, other methods for preparing and screening the antibodies that change are also available to the skilled person.

Phagemid display technology provides a powerful tool for generating and selecting novel proteins that bind to ligands such as antigens. Using phagemid display technology allows for the generation of large libraries of protein variants that can be rapidly sorted for sequences that bind to target molecules with high affinity. Nucleic acids encoding variant polypeptides are typically fused to nucleic acid sequences encoding viral coat proteins, such as gene III or gene VIII proteins. Monovalent phagemid display systems have been developed in which nucleic acid sequences encoding proteins or polypeptides are fused to nucleic acid sequences encoding a portion of gene III protein. (Bass, S., Proteins, 8:309 (1990); Lowman and Wells, Methods: A Companion to Methods in Enzymology, 3:205 (1991)). In monovalent phagemid display systems, the gene fusion is expressed at low levels, while the wild-type gene III protein is also expressed, so that the infectivity of the (viral) particle is retained. Methods of generating peptide libraries and screening those libraries have been disclosed in numerous patents (eg, US Pat. No. 5,723,286, US Pat. No. 5,432,018, US Pat. No. 5,580,717, US Pat. No. 5,427,908, and US Pat. No. 5,498,530).

The library of antibody or antigen-binding polypeptide has been prepared by many methods (comprising by inserting random DNA sequence and changing single gene or by cloning the family of related gene).The method using phage (grain) display to show antibody or antigen-binding fragment has been described in U.S. Patent number 5,750,373,5,733,743,5,837,242,5,969,108,6,172,197,5,580,717 and 5,658,727.Then screening library obtains the expression of antibody or antigen-binding protein with desired characteristic.

Methods for substituting selected amino acids into template nucleic acids are well established in the art, and some such methods are described herein. For example, hypervariable region residues can be substituted using the Kunkel method. See, for example, Kunkel et al., Methods Enzymol. 154:367-382 (1987).

The sequence of the oligonucleotide includes one or more designed codon sets for changing the hypervariable region residues. A codon set is a set of triplet sequences of different nucleotides encoding the desired variant amino acid. A codon set can be represented by symbols referring to specific nucleotides or equimolar mixtures of nucleotides as given below according to the IUB code.

IUB coding

Oligonucleotide or primer sets can be synthesized by standard methods.For example, by solid phase synthesis, one group of oligonucleotide is synthesized, it contains all possible combinations of the nucleotide triplet that represents being provided by the codon set and will encode the amino acid of expectation group.It is well known in the art to synthesize oligonucleotide with selected nucleotide " degeneracy " on specific position.This oligonucleotide group with definite codon set can use the nucleic acid synthesizer (purchased from, for example, Applied Biosystems, Foster City, CA) that purchases to synthesize, or directly purchase and obtain (for example, purchased from Life Technologies, Rockville, MD).Therefore, the synthetic oligonucleotide group with specific codon set will comprise a large amount of oligonucleotide with different sequences usually, and the difference of wherein said sequence is set up by the codon set in the whole sequence.Oligonucleotide as used in the present invention has the sequence that can hybridize with the variable domain nucleic acid template, and can also comprise the restriction enzyme site that is used to clone purpose.

In one method, a nucleic acid sequence encoding a variant amino acid is generated using oligonucleotide-mediated mutagenesis. This technique is well known in the art and is described, for example, in Zoller et al., Nucleic Acids Res. 10:6487-6504 (1987). Briefly, a nucleic acid sequence encoding a variant amino acid is generated by hybridizing an oligonucleotide set encoding the desired codon set with a DNA template, wherein the DNA template is a plasmid containing a single-stranded form of the variable region nucleic acid template sequence. Following hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template, which will thereby incorporate the oligonucleotide primer and will contain the codon set provided by the oligonucleotide set.

Typically, oligonucleotides of at least 25 nucleotides in length are used. The best oligonucleotides have 12-15 nucleotides that are fully complementary to the template on either side of the encoding mutation nucleotide. This ensures that the oligonucleotide will properly hybridize with the single-stranded DNA template molecule. Oligonucleotides are easily synthesized using techniques known in the art, such as Crea et al., Proc. Nat'l. Acad. Sci. USA (Proceedings of the National Academy of Sciences of the United States of America), 75:5765 (1978) described methods.

DNA templates are prepared using vectors derived from bacteriophage M13 vectors (commercially available M13mp18 and M13mp19 vectors are suitable), or vectors containing a single-stranded phage origin of replication as described by Viera et al., Meth. Enzymol., 153:3 (1987). Thus, the DNA to be mutated is inserted into one of these vectors to prepare a single-stranded template. Preparation of single-stranded templates is described in Sambrook et al., supra, Sections 4.21-4.41.

In order to change the native DNA sequence, the oligonucleotide is hybridized with the single-stranded template under suitable hybridization conditions. Then add a DNA polymerase, usually the Klenow fragment of T7 DNA polymerase or DNA polymerase I, use the oligonucleotide as a synthetic primer, the complementary chain of the synthetic template. Therefore, a heteroduplex molecule is formed so that one chain of DNA encodes the gene 1 of the mutant form, and the other chain (starting template) encodes the gene 1 of the natural, unchanged sequence. Then the heteroduplex molecule is transformed into a suitable host cell, usually a prokaryotic organism, for example in the intestinal bacteria E.coli JM101. After cultivating the cell, the cell is spread on an agar plate and screened with a radioactive isotope phosphorus ^32- labeled oligonucleotide primer to determine the bacterial clone containing the mutant DNA.

The method just described above can be improved to generate homoduplex molecules, wherein both chains of the plasmid contain mutations. The changes are as follows: the single-stranded oligonucleotide is annealed to the single-stranded template as described above. A mixture of three deoxyribonucleic acids, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP) and deoxyribothymidine (dTT) is combined with a modified thiodeoxyribocytidine known as dCTP-(aS) (available from Amersham). This mixture is added to the template-oligonucleotide complex. When DNA polymerase is added to this mixture, a DNA chain identical to the template is generated except that the mutated bases. In addition, this new DNA chain contains dCTP-(aS), rather than dCTP, which prevents the chain from being digested by restriction endonucleases. On the template chain of the double-stranded heteroduplex, after a gap is generated with a suitable restriction enzyme, the template chain is digested with ExoIII nuclease or another suitable nuclease in a region different from the site to be mutated. The reaction is then terminated, leaving the molecule with only a partially single strand. In the presence of all four deoxyribonucleoside triphosphates, ATP, and DNA ligase, DNA polymerase is used to produce a complete double-stranded DNA homoduplex. This homoduplex molecule is then transformed into a suitable host cell.

As previously noted, the sequence length of the oligonucleotide set is sufficient to hybridize to the template nucleic acid and also, but not necessarily, contain restriction sites. DNA templates can be prepared using vectors derived from bacteriophage M13 vectors, or vectors containing single-stranded phage origins of replication as described by Viera et al., Meth. Enzymol., 153:3 (1987). Therefore, the DNA to be mutated must be inserted into one of these vectors to prepare a single-stranded template. The preparation of single-stranded templates is described in Sambrook et al., supra, sections 4.21-4.41.

According to another method, prepare library by upstream and downstream oligonucleotide groups, each oligonucleotide group has the different oligonucleotides of a large amount of sequences, and different sequences are determined by providing codon sets in the oligonucleotide sequences. Variable region template nucleic acid sequence and upstream and downstream oligonucleotide groups can be used for polymerase chain reaction, prepare " library " of PCR product. PCR product can be referred to as " nucleic acid expression cassette ", and this is owing to use the molecular biology technique that has been established, they can be fused with other relevant or irrelevant nucleic acid sequences, for example, viral coat protein and dimerization domain.

The sequences of the PCR primers include one or more codon sets designed for solvent accessible and highly variable positions in the hypervariable region. As described above, a codon set is a set of different nucleotide triplet sequences encoding the desired variant amino acids. Antibody selections that meet the desired criteria (e.g., selected by appropriate screening/selection steps) can be isolated and cloned using standard recombinant techniques.

B. Recombinant Preparation

The present invention also provides isolated nucleic acids encoding anti-PD-L1 antibodies, vectors and host cells comprising such nucleic acids, and recombinant techniques for producing the antibodies.

In order to recombinantly produce an antibody, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (DNA amplification) or expression. Conventional procedures can be used to easily separate the DNA encoding the monoclonal antibody and sequence it (e.g., using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of the antibody). Many vectors are available. The selection of the vector depends in part on the host cell to be used. Typically, preferred host cells are prokaryotic or eukaryotic (usually mammalian) in origin.

1. Antibody production in prokaryotic cells

a) Vector construction

Standard recombinant techniques can be used to obtain the polynucleotide sequences encoding the antibody polypeptide components of the present invention. The desired polynucleotide sequence can be isolated and sequenced from antibody-producing cells such as hybridoma cells. Alternatively, a nucleotide synthesizer or PCR technology can be used to synthesize polynucleotides. Once obtained, the sequence encoding the polypeptide is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. For the present invention, many vectors available and known in the art can be used. The selection of suitable vectors will depend primarily on the size of the nucleic acid to be inserted into the vector and the specific host cell to be transformed with the vector. According to its function (amplification or expression of heterologous polynucleotides, or both) and its compatibility with the specific host cell in which it resides, every kind of vector contains a variety of components. Vector components typically include, but are not limited to, an origin of replication, a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, a heterologous nucleic acid insert, and a transcription termination sequence.

In general, the plasmid vectors used with host cells comprise replicons and control sequences derived from species compatible with these hosts. The vectors typically carry replication sites and marker sequences that can provide phenotypic selection in transformed cells. For example, E. coli is typically transformed with the plasmid pBR322 derived from an E. coli species. pBR322 comprises genes encoding ampicillin (Amp) and tetracycline (Tet) resistance, thereby providing a means for easily identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or phages may also comprise or be modified to comprise a promoter that can be used by microbial organisms to express endogenous proteins. Carter et al., U.S. Patent No. 5,648,237, describe in detail examples of pBR322 derivatives for expressing specific antibodies.

In addition, phage vectors containing replicons and control sequences compatible with the host microorganism can be used as transformation vectors for these hosts. For example, phages such as GEM.TM.-11 can be used to construct recombinant vectors that can be used to transform susceptible host cells such as E. coli LE392.

The expression vectors of the present invention may contain two or more promoter-cistron pairs encoding each polypeptide component. A promoter is an untranslated regulatory sequence located upstream (5') of a cistron that regulates its expression. Prokaryotic promoters are generally divided into two categories, inducible and constitutive. Inducible promoters are promoters that initiate increased levels of transcription of the cistron under their control in response to changes in culture conditions (e.g., the presence or absence of nutrients or temperature).

A large number of promoters that are recognized by various potential host cells are well known. By cutting out the promoter in the source DNA through restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present invention, the promoter of selection can be operably connected to the cistron DNA encoding the light chain or heavy chain. Natural promoter sequences and many heterologous promoters can be used to direct the amplification and/or expression of the target gene. In some embodiments, heterologous promoters are used because they generally allow higher transcription and higher yield of the expressed target gene compared to the natural target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the galactamase and lactose promoter systems, the tryptophan (trp) promoter system, and hybrid promoters such as the tac or trc promoters. However, other promoters that are functional in bacteria (such as other known bacterial or phage promoters) are also suitable. Their nucleotide sequences have been published, allowing the skilled worker to operably link them to the cistrons encoding the target light and heavy chains using linkers or adapters providing any desired restriction sites (Siebenlist et al. (1980) Cell 20 : 269).

In one aspect, each cistron in the recombinant vector comprises the secretion signal sequence component that instructs the expressed polypeptide to cross the membrane and transport. Generally speaking, the signal sequence can be the component of the vector, or it can be a part for the target polypeptide DNA of the insertion vector. The signal sequence selected for the present invention should be the signal sequence that is subject to host cell recognition and processes (i.e., excision by signal peptidase). For the prokaryotic host cell that does not recognize and processes the natural signal sequence of heterologous polypeptide, the signal sequence is substituted with the prokaryotic signal sequence of the group that is for example made up of the following: alkaline phosphatase, penicillinase, Ipp or heat-stable enterotoxin II (STII) leader sequence, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of the invention, the signal sequence all used in two cistrons of the expression system is the STII signal sequence or its variant.

On the other hand, production of immunoglobulins according to the present invention can occur in the cytoplasm of the host cell, thus eliminating the need for a secretion signal sequence within each cistron. In this case, the immunoglobulin light and heavy chains are expressed, folded, and assembled within the cytoplasm to form functional immunoglobulins. Certain host strains (e.g., trxB ^- strains of E. coli) provide cytoplasmic conditions that favor disulfide bond formation, thereby allowing for proper folding and assembly of the expressed protein subunits. Proba and Pluckthun, Gene, 159 :203 (1995).

The present invention provides an expression system that can regulate the quantitative ratio of expressed polypeptide components to maximize the yield of secreted and correctly assembled antibodies of the present invention. This regulation is achieved at least in part by simultaneously regulating the translational strength of the polypeptide components. Simmons et al., U.S. Patent No. 5,840,523 discloses a technique for regulating translational strength. It utilizes variants of the translation initiation region (TIR) within a cistron. For a given TIR, a series of amino acid or nucleotide sequence variants with a certain range of translational strengths can be created, thereby providing a convenient means of regulating this factor for the desired expression level of a particular chain. TIR variants can be generated by conventional mutagenesis techniques to cause codon changes that can alter the amino acid sequence (although silent changes in the nucleotide sequence are preferred). Changes in the TIR can include, for example, changes in the number or spacing of Shine-Dalgarno sequences, and changes in the signal sequence. One method for generating mutant signal sequences is to generate a "codon library" at the beginning of the coding sequence that does not alter the amino acid sequence of the signal sequence (i.e., the changes are silent). This can be achieved by changing the third nucleotide position of each codon; in addition, some amino acids, such as leucine, serine, and arginine, have multiple first and second positions, which can increase the complexity in library construction. This mutagenesis method is described in detail in Yansura et al. (1992) METHODS: A Companion to Methods in Enzymol. 4: 151-158.

Preferably, for each cistron in the vector, a set of vectors with a range of TIR strengths is generated. This limited set provides a comparison of the expression level of each chain and the yield of the desired antibody product under various TIR strength combinations. TIR strength can be measured by quantifying the expression level of a reporter gene, as described in detail in Simmons et al., U.S. Patent 5,840,523. Based on the comparison of translational strengths, the desired individual TIRs are selected for combination in the expression vector constructs of the present invention.

b) Prokaryotic host cells

Prokaryotic host cells suitable for expressing antibodies of the present invention include archaebacteria and eubacteria, such as gram-negative or gram-positive organisms. Examples of useful bacteria include Escherichia (Escherichia) (such as Escherichia coli (E.coli)), Bacillus (Bacilli) (such as Bacillus subtilis (B.subtilis)), Enterobacteria (Enterobacteria), Pseudomonas species (Pseudomonas species) (such as Pseudomonas aeruginosa (P.aeruginosa)), Salmonella typhimurium (Salmonella typhimurium), Serratia marcescans (Serratia marcescans), Klebsiella (Klebsiella), Proteus (Proteus), Shigella (Shigella), Rhizobium (Rhizobia), Vitreoscilla (Vitreoscilla) or Paracoccus (Paracoccus). In one embodiment, gram-negative cells are used. In one embodiment, E. coli cells are used as hosts of the present invention. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology , vol. 2 (Washington,

DC: American Society for Microbiology (American Society for Microbiology, 1987), pp. 1190-1219; ATCC Accession No. 27,325) and derivatives thereof, including strain 33D3, which has the genotype W3110ptr3lacIqlacL8(nmpc-fepE) ^degP41kanR (U.S. Patent No. 5,639,635). Other strains and derivatives thereof, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli 1776 (ATCC 31,537) and E. coli RV308 (ATCC 31,608) are also suitable. These examples are illustrative and non-limiting. Methods for constructing derivatives of any of the above-mentioned bacteria with a specified genotype are known in the art and are described in, for example, Bass et al., Proteins , 8 : 309-314 (1990). It is generally necessary to consider the replicability of the replicon in the bacterial cell to select an appropriate bacterium. For example, E. coli, Serratia, or Salmonella species may be suitably used as hosts when well-known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to provide the replicon.

Generally, the host cells should secrete minimal amounts of proteolytic enzymes, and it may be desirable to incorporate additional protease inhibitors into the cell culture.

c) Antibody production

Host cells are transformed with the above-mentioned expression vectors and cultured in conventional nutrient media that have been appropriately modified to induce promoters, select transformants, or amplify genes encoding the desired sequence. Transformation involves importing DNA into prokaryotic hosts so that the DNA can replicate, either as an extrachromosomal element or through chromosomal components. Depending on the host cell used, standard techniques suitable for these cells are used for transformation. Calcium treatment using calcium chloride is commonly used for bacterial cells with a strong cell wall barrier. Another method of transformation employs polyethylene glycol/DMSO. Electroporation is also a technique employed.

Prokaryotic cells for producing antibodies of the present invention are cultured in a culture medium known in the art and suitable for culturing selected host cells. Examples of suitable culture media include LB medium (luria broth) supplemented with essential nutrient supplements. In some embodiments, the culture medium also contains a selection agent selected based on the construction of the expression vector to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the culture medium for culturing cells expressing an ampicillin resistance gene.

In addition to carbon, nitrogen and inorganic phosphate sources, any necessary supplements may be present at appropriate concentrations, either alone or as a mixture with another supplement or medium, such as a complex nitrogen source. Optionally, the medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycollate, dithioerythritol and dithiothreitol.

Prokaryotic host cells are cultured at a suitable temperature. For example, for culturing E. coli, a temperature range of about 20° C. to about 39° C. is preferred, more preferably about 25° C. to about 37° C., and even more preferably about 30° C. is preferred. Depending primarily on the host organism, the pH of the culture medium can be any pH in the range of about 5 to about 9. For E. coli, the pH is preferably about 6.8 to about 7.4, more preferably about 7.0.

If an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for activating the promoter. In one aspect of the present invention, the PhoA promoter is used to control transcription of the polypeptide. Thus, for induction, the transformed host cells are cultured in a phosphate-limited medium. Preferably, the phosphate-limited medium is C.R.A.P medium (see, e.g., Simmons et al., J. Immunol. Methods (2002), 263:133-147). Depending on the vector construct employed, a variety of other inducers may be employed, as is known in the art.

The expressed antibody protein of the present invention is secreted into the periplasm of the host cell and recovered therefrom. Protein recovery generally involves destroying the microorganism, usually by means such as osmotic shock, ultrasonic treatment or lysis. Once the cells are destroyed, cell debris or whole cells can be removed by centrifugation or filtration. The protein can be further purified by, for example, affinity resin chromatography. Alternatively, the protein may be transported to and separated from the culture medium. The cells can be removed from the culture medium, and the culture supernatant can be filtered and concentrated for further purification of the generated protein. The expressed polypeptide can be further isolated and identified using generally known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot analysis.

In another embodiment, antibody production is carried out in a large number by fermentation process.Multiple large-scale fed-batch fermentation processes can be used for producing recombinant protein.Large-scale fermentation has the capacity of at least 1000 liters, preferably the capacity of about 1,000 to 100,000 liters.These fermentation tanks use agitator impellers to distribute oxygen and nutrient, especially glucose (preferred carbon source/energy source).Small-scale fermentation generally refers to the fermentation carried out in the fermentation tank that volume capacity is no more than about 100 liters, and scope can be about 1 liter to about 100 liters.

In the fermentation process, induction of protein expression is typically initiated after the cells have been cultured under appropriate conditions to a desired density (e.g., an OD ₅₅₀ of about 180-220, at which stage the cells are in the early stationary phase). Depending on the vector construct employed, a variety of inducers can be used, as known in the art and described above. The cells may be cultured for shorter periods of time prior to induction. Typically, the cells are induced for about 12-50 hours, but longer or shorter induction times may be used.

To improve the yield and quality of the antibodies of the present invention, various fermentation conditions can be modified. For example, to improve the correct assembly and folding of the secreted antibody polypeptide, an additional vector that overexpresses chaperone proteins such as the Dsb proteins (DsbA, DsbB, DsbC, DsbD, and/or DsbG) or FkpA (a peptidylproline cis-trans isomerase with chaperone activity) can be used to co-transform the host prokaryotic cells. Chaperone proteins have been shown to promote the correct folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J Bio Chem 274:19601-19605; Georgiou et al., U.S. Patent No. 6,083,715; Georgiou et al., U.S. Patent No. 6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol. Microbiol. 39:199-210.

In order to minimize proteolysis of expressed heterologous proteins, especially those that are sensitive to proteolysis, certain host strains deficient in proteolytic enzymes can be used in the present invention. For example, the host cell strain can be modified to include genetic mutations in genes encoding known bacterial proteases, such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI, and combinations thereof. Some E. coli protease-deficient strains are available, see, for example, Joly et al. (1998), supra; Georgiou et al., U.S. Patent No. 5,264,365; Georgiou et al., U.S. Patent No. 5,508,192; Hara et al., Microbial Drug Resistance, 2 : 63-72 (1996).

In the expression system encoding the antibodies of the present invention, E. coli strains deficient in proteolytic enzymes and transformed with plasmids overexpressing one or more chaperone proteins can be used as host cells.

d) Antibody purification

The antibody proteins generated herein are further purified to obtain substantially homogeneous products for further determination and use. Standard protein purification methods known in the art can be used. The following process is illustrative of suitable purification processes: fractionation on an immunoaffinity or ion exchange column, ethanol precipitation, reversed-phase HPLC, chromatography on a silica or cation exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75.

In one aspect, protein A immobilized on a solid phase is used for immunoaffinity purification of the full-length antibody product of the present invention. Protein A is a 41 kD cell wall protein from Staphylococcus aureas that binds to the Fc region of antibodies with high affinity. Lindmark et al. (1983) J. Immunol. Meth. 62:1-13. The solid phase to which protein A is immobilized is preferably a column having a glass or quartz surface, more preferably a controlled pore glass column or a silicic acid column. In some applications, the column is coated with a reagent such as glycerol in an attempt to prevent nonspecific adhesion of contaminants. The solid phase is then washed to remove contaminants that are nonspecifically bound to the solid phase. Finally, the antibody of interest is recovered from the solid phase by elution.

2. Antibody production in eukaryotic cells

For eukaryotic expression, vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

a) Signal sequence components

Vectors used in eukaryotic hosts may also contain an insertion encoding a signal sequence or other polypeptide with a specific cleavage site at the N-terminus of the mature protein or polypeptide. Preferably, the heterologous signal sequence selected is one that is recognized and processed by the host cell (i.e., cleaved by a signal peptidase). For mammalian cell expression, mammalian signal sequences and viral secretion leaders, such as the herpes simplex virus gD signal, can be used.

The DNA for these precursor regions is ligated in frame to the DNA encoding the antibody of the present invention.

b) Origin of replication

Typically, mammalian expression vectors do not require an origin of replication component (the SV40 origin may often be used only because it contains the early promoter).

c) Selection of genetic components

Expression and cloning vectors may contain a selection gene, also known as a selectable marker. Typical selection genes encode proteins that: (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) complement corresponding auxotrophic deficiencies; or (c) provide critical nutrients that cannot be obtained from complex media, such as the gene encoding D-alanine racemase from Bacilli.

One example of a selection scheme utilizes a drug to retard the growth of host cells. Those cells that are successfully transformed with the heterologous gene produce a protein that confers drug resistance and are therefore spared the selection scheme. Examples of this type of dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.

Another example of a suitable selectable marker for mammalian cells is one that allows identification of cells competent to take up nucleic acid encoding an antibody of the invention, such as DHFR, thymidine kinase, metallothionein I and II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, and the like.

For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a medium containing methotrexate (Mtx, a competitive antagonist of DHFR). When wild-type DHFR is employed, a suitable host cell is a Chinese hamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

Alternatively, host cells (particularly wild-type hosts containing endogenous DHFR) transformed or co-transformed with DNA sequences encoding an antibody, wild-type DHFR protein, and another selectable marker, such as aminoglycoside 3'-phosphotransferase (APH), can be selected by culturing the cells in a medium containing a selection agent for the selectable marker, such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199.

d) Promoter component

Expression and cloning vectors usually comprise a promoter that is recognized by the host organism and are operably connected to the nucleic acid encoding the desired antibody sequence. In fact, all eukaryotic genes have an AT-rich region, which is located approximately 25 to 30 bases upstream of the site where transcription begins. Another sequence found 70 to 80 bases upstream of the transcription start site of many genes is the CNCAAT region, where N can be any nucleotide. 3' ends in most eukaryotic vectors are AATAAA sequences, which may be signals that add polyadenylic acid (polyA) tails to the 3' ends of the coding sequence. All of these sequences can be inserted into eukaryotic expression vectors.

Other promoters suitable for use with eukaryotic hosts include the phoA promoter, beta-lactamase and lactose promoter systems, alkaline phosphatase promoter, tryptophan (trp) promoter systems, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are suitable. Promoters used in bacterial systems will also contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the antibody polypeptide.

Transcription of the antibody polypeptide from the vector in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus type 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and most preferably Simian Virus 40 (SV40); from heterologous mammalian promoters (e.g., the actin promoter or the immunoglobulin promoter); or from heat shock promoters, provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtained in the form of an SV40 restriction fragment, which also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained in the form of a HindIII E restriction fragment. A system for expressing DNA in a mammalian host using bovine papilloma virus as a vector is disclosed in U.S. Patent No. 4,419,446. A modification of this system is described in U.S. Patent No. 4,601,978. Regarding the expression of human interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus, see also Reyes et al., Nature 297:598-601 (1982). Alternatively, the Rous sarcoma virus long terminal repeat sequence can be used as a promoter.

e) Enhancer element components

Often, the transcription of the DNA encoding the antibody of the present invention by higher eukaryotic cells is increased by inserting an enhancer sequence into the vector. Many enhancer sequences from mammalian genes (globulin, elastase, albumin, α-fetoprotein, and insulin) are now known. However, enhancers from eukaryotic cell viruses are commonly used. Examples include the enhancer on the late side of the SV40 replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the enhancer on the late side of the polyoma replication origin, and the adenovirus enhancer. For enhancing elements that activate eukaryotic promoters, see also Yaniv, Nature 297:17-18 (1982). The enhancer can be spliced into the vector and located at the 5' or 3' position of the antibody coding sequence, but is preferably located at the 5' site of the promoter.

f) Transcription termination component

Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) also contain sequences necessary for terminating transcription and stabilizing the mRNA. Such sequences are typically available from the 5' and occasionally 3' ends of the untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed into polyadenylated fragments in the untranslated region of the mRNA encoding the antibody. A useful transcription termination component is the bovine growth hormone polyadenylation region. See WO 94/11026 and the expression vectors disclosed therein.

g) Selection and transformation of host cells

Suitable host cells for cloning or expressing the DNA in the vectors herein include higher eukaryotic cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651), human embryonic kidney line (293 cells or 293 cells subcloned for suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1, ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL 1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)), MRC 5 cells, FS4 cells and human hepatoma line (Hep G2).

For antibody production, host cells are transformed with the expression or cloning vectors described above and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the gene encoding the desired sequence. Examples of useful mammalian host cell lines are

h) Host cell culture

Host cells for producing the antibodies of the present invention can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing host cells. In addition, any culture medium described in the following literature can be used as a culture medium for host cells: Ham et al., Meth. Enz. 58: 44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Patent Reissue 30,985. Any of these media may be supplemented as needed with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN ^™ ), trace elements (defined as inorganic compounds typically present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements known to those skilled in the art may also be included at appropriate concentrations. The culture conditions, such as temperature, pH, and the like, will be those previously used for the host cell selected for expression and will be apparent to those of ordinary skill in the art.

i) Antibody purification

When using recombinant technology, antibodies can be generated in the cell or periplasmic space, or directly secreted into the culture medium. If the antibody is generated in the cell, it is first necessary to remove particulate debris, such as host cells or cleavage fragments, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10 : 163-167 (1992) have described the procedure for separating antibodies, which are secreted into the periplasmic space of Escherichia coli. In short, the cell paste melts in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) in about 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the culture medium, then usually first commercial protein concentration filters (such as Amicon or Millipore Pellicon ultrafiltration units) are used to concentrate the supernatant from these expression systems. Protease inhibitors such as PMSF can be included in any of the above steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

The antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, which is a preferred purification technique. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human immunoglobulins containing 1, 2, or 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and people 3 (Guss et al., EMBO J. 5: 1567-1575 (1986)). The most commonly used matrix to which the affinity ligand is attached is agarose, but other matrices can be used. Physically stable matrices such as controlled pore glass or poly (styrene-divinyl) benzene can obtain faster flow rates and shorter processing times than agarose. If the antibody contains a _CH3 domain, it can be purified using Bakerbond ABX ^™ resin (JT Baker, Phillipsburg, NJ). Other protein purification techniques such as fractionation on an ion exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE ^™ , chromatography on anion or cation exchange resins (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation can also be used, depending on the antibody to be recovered.

Following any preliminary purification steps, the mixture containing the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer having a pH of about 2.5-4.5, preferably at a low salt concentration (e.g., about 0-0.25 M salt).

C. Antibody Preparation

1) Polyclonal antibodies

Polyclonal antibodies are typically raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to couple the relevant antigen to a protein that is immunogenic in the species to be immunized (e.g., keyhole limpet hemocyanin (KLH), serum albumin, _bovine thyroglobulin, or soybean ^trypsin inhibitor) using bifunctional or derivatizing agents such as maleimidobenzoylsulfosuccinimide ester (conjugated through cysteine residues), N-hydroxysuccinimide (conjugated through lysine residues), glutaraldehyde, succinic anhydride, SOCl 2 , or R 1 N═C═NR (wherein R and R ¹ are independently lower alkyl). Examples of adjuvants that can be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). One skilled in the art can select an immunization regimen without undue experimentation.

Animals are immunized against antigens, immunogenic conjugates, or derivatives by mixing, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with 1/5-1/10 of the initial amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7-14 days, the blood of the animals is collected and the antibody titer of the serum is determined. The animals are boosted until the titer reaches a plateau. Conjugates can also be prepared as protein fusions in recombinant cell culture. Similarly, coagulants (such as alum) are suitable for enhancing the immune response.

2) Monoclonal antibodies

Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of dispersed antibodies.

For example, monoclonal antibodies can be made using the hybridoma method first described by Kohler et al., Nature, 256 :495 (1975), or can be made by recombinant DNA methods (US Pat. No. 4,816,567).

In the hybridoma method, a mouse or other suitable host animal (such as a hamster) is immunized as described above to stimulate lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent (such as polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1986).

The immunizing agent generally includes an antigenic protein or a fusion variant thereof. Peripheral blood lymphocytes ("PBLs") (if cells of human origin are desired) or spleen cells or lymph node cells (if non-human mammalian origin is desired) are typically used. The lymphocytes are then fused with an immortalized cell line using an appropriate fusing agent (such as polyethylene glycol) to form a hybridoma cell. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103.

Immortalized cell line is typically the mammalian cell of transformation, particularly the myeloma cell of rodent, cattle or people's origin.Usually, rat or mouse myeloma cell line is used.The hybridoma cell of so preparing is inoculated and cultivated in suitable culture medium, and described culture medium preferably contains one or more substances that suppress the growth or survival of the parent myeloma cell that does not fuse.For example, if the parent myeloma cell lacks enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), then the culture medium for hybridoma typically will contain hypoxanthine, aminopterin and thymidine (HAT culture medium), and these substances stop HGPRT defective cell growth.

Preferred immortalized myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these myeloma cells, preferred are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center (San Diego, California, USA) and SP-2 cells (and derivatives thereof, e.g., X63-Ag8-653) available from the American Type Culture Collection (Manassas, Virginia, USA). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol. 133 :3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York, 1987).

Culture medium in which hybridoma cells are growing is assayed for the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The presence of monoclonal antibodies against the desired antigen in the culture medium of the hybridoma cells can be determined. Preferably, the binding affinity and specificity of the monoclonal antibodies can be determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked assays (ELISA). Such techniques and assays are known in the art. For example, binding affinity can be determined using the Scatchard analysis of Munson et al., Anal. Biochem. 107 :220 (1980).

After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and/or activity, the clones can be subcloned by limiting dilution methods and cultured by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. Alternatively, hybridoma cells can be cultured in vivo as tumors in mammals.

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures (such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography).

Monoclonal antibodies can also be prepared by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567 and described above. The DNA encoding the monoclonal antibody is easy to separate and sequenced using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to genes encoding mouse antibody heavy and light chains). Hybridoma cells can serve as a preferred source of such DNA. Once separated, the DNA can be placed into an expression vector, which is then transfected into a host cell (such as E. coli (E. coli) cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells) that does not otherwise generate immunoglobulin proteins to obtain the synthesis of monoclonal antibodies in such recombinant host cells. Review articles on recombinantly expressed DNA encoding antibodies in bacteria include Skerra et al., Curr. Opinion in Immunol., 5 : 256-262 (1993) and Plückthun, Immunol. Revs. 130 : 151-188 (1992).

In another embodiment, antibodies can be isolated from phage antibody libraries constructed using the techniques described in McCafferty et al., Nature 348 : 552-554 (1990). Clackson et al., Nature 352 : 624-628 (1991) and Marks et al., J. Mol. Biol. 222 : 581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications documented the generation of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio/Technology 10 : 779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nucl. Acids Res. 21 : 2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

The DNA can also be modified, for example, by replacing the homologous murine sequences with coding sequences for human heavy and light chain constant domains (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81 : 6851 (1984)), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. Typically, such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-binding site of an antibody to create a chimeric bivalent antibody comprising one antigen-binding site specific for an antigen and another antigen-binding site specific for a different antigen.

The monoclonal antibodies described herein can be monovalent antibodies, and methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinantly expressing immunoglobulin light chains and modified heavy chains. The heavy chains are typically truncated at any point in the Fc region to avoid cross-linking of the heavy chains. Alternatively, the relevant cysteine residues are replaced or deleted by other amino acid residues to avoid cross-linking. In vitro methods are also suitable for preparing monovalent antibodies. Antibodies can be digested using conventional techniques known in the art to produce fragments thereof, particularly Fab fragments.

Chimeric or hybrid antibodies can also be prepared in vitro using methods known in synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins can be constructed using disulfide-exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

3) Humanized antibodies

Antibodies of the present invention can also include humanized or human antibodies. The humanized form of non-human (e.g., mouse) antibodies is a chimeric immunoglobulin, immunoglobulin chain, or its fragment (e.g., Fv, Fab, Fab', F(ab') ₂ , or other antigen-binding subsequences (subsequences) of an antibody) comprising a minimal sequence derived from a non-human immunoglobulin. Humanized antibodies include human immunoglobulins (receptor antibodies) wherein residues from the complementarity determining regions (CDRs) (e.g., HVRs) of the receptor are replaced with residues from the CDRs of non-human species (donor antibodies) such as mice, rats, or rabbits with desired specificity, avidity, and ability. In some cases, the Fv framework residues of human immunoglobulins are replaced with corresponding non-human residues. Humanized antibodies can also include residues not found in receptor antibodies or the CDRs or framework sequences introduced. In general, a humanized antibody will comprise substantially all of at least one, typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are FR regions of a human immunoglobulin consensus sequence. The humanized antibody will optimally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Jones et al., Nature 321 : 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2 : 593-596 (1992).

Methods for humanizing non-human antibodies are well known in the art. Typically, a humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from the "import" variable domain. Humanization can essentially follow the methods of Winter and colleagues (Jones et al., Nature 321 : 522-525 (1986); Riechmann et al., Nature 332 : 323-327 (1988); Verhoeyen et al., Science 239 : 1534-1536 (1988)), or by replacing the corresponding sequences of a human antibody with rodent CDRs or CDR sequences. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), in which substantially less than the entire human variable domain is replaced with the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

The selection of human variable domains (including both light and heavy chains) for constructing humanized antibodies is very important for reducing antigenicity. According to the so-called "best-fit" method, the entire library of known human variable domain sequences is screened with the variable domain sequences of rodent antibodies. The human sequence closest to the rodent is then accepted as the human framework (FR) of the humanized antibody (Sims et al., J. Immunol. 151 : 2296 (1993); Chothia et al., J. Mol. Biol. 196 : 901 (1987)). Another method uses a specific framework derived from the consensus sequence of all human antibodies of a specific light or heavy chain subgroup. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad Sci. USA 89 :4285 (1992); Presta et al., J. Immunol. 151 :2623 (1993)).

More importantly, the antibody maintains high affinity for antigens and other favorable biological properties after humanization. To achieve this goal, according to a preferred method, humanized antibodies are prepared by using a three-dimensional model of the parent and humanized sequences to analyze the parent sequence and various conceptual humanized products. Three-dimensional immunoglobulin models are publicly available and are familiar to those skilled in the art. Computer programs that illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences can be obtained. Checking these displayed images allows analysis of the possible effects of residues in the functioning of the candidate immunoglobulin sequence, i.e., analyzing the residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected from the receptor and the input sequence and combined to obtain desired antibody characteristics, such as increased avidity for the target antigen. Generally speaking, the direct and most substantial involvement of CDR residues affects the binding to the antigen.

Various forms of humanized antibodies are contemplated. For example, the humanized antibody can be an antibody fragment, such as a Fab, which is optionally conjugated to one or more cytotoxic agents to produce an immunoconjugate. Alternatively, the humanized antibody can be a complete antibody, such as a complete IgG1 antibody.

4) Human antibodies

As an alternative to humanization, human antibodies can be produced. For example, it is now possible to generate transgenic animals (e.g., mice) that are able to generate a full repertoire of human antibodies after immunization in the absence of endogenous immunoglobulin production. For example, homozygous deletion of the antibody heavy chain joining region ( _JH ) gene in chimeric and germline mutant mice has been documented to result in complete inhibition of endogenous antibody production. Transferring the human germline immunoglobulin gene array in such germline mutant mice will result in the generation of human antibodies after antigen challenge. See, for example, Jakobovits et al., Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA) 90 : 2551 (1993); Jakobovits et al., Nature 362 : 255-258 (1993); Bruggermann et al., Year in Immuno. 7 : 33 (1993); U.S. Patent No. 5,591,669 and WO 97/17852.

Alternatively, phage display technology can be used to produce human antibodies and antibody fragments in vitro from a repertoire of immunoglobulin variable (V) domain genes from unimmunized donors. McCafferty et al., Nature 348 : 552-553 (1990); Hoogenboom and Winter, J. Mol. Biol. 227 : 381 (1991). According to this technology, antibody V domain genes are cloned in a frame-matched manner into the major or minor coat protein gene of a filamentous phage (such as M13 or fd) and displayed as functional antibody fragments on the surface of the phage particle. Because filamentous phage particles contain a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in the selection of genes encoding antibodies that display those properties. In this way, the phage mimics some of the properties of B cells. Phage display can be performed in a variety of formats, as reviewed in, for example, Johnson, Kevin S. and Chiswell, David J., Curr Opin in Struct Biol 3 :564-571 (1993). Several sources of V gene segments can be used for phage display. Clackson et al., Nature 352:624-628 (1991) isolated a large and diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. By essentially following the techniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991) or Griffith et al., EMBO J. 12:725-734 (1993), it is possible to construct a repertoire of V genes from unimmunized human donors and isolate antibodies to a wide variety of antigens, including self-antigens. See also U.S. Patent Nos. 5,565,332 and 5,573,905.

The techniques of Cole et al. and Boerner et al. can also be used to prepare human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol. 147 (1): 86-95 (1991)). Similarly, human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals, such as mice, in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, production of human antibodies is observed that closely resembles that seen in humans in all aspects, including gene rearrangement, assembly, and antibody repertoire. This method is described, for example, in U.S. Patents 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10 :779-783 (1992); Lonberg et al., Nature 368 :856-859 (1994); Morrison, Nature, 368 :812-13 (1994); Fishwild et al., Nature Biotechnology 14 :845-51 (1996); Neuberger, Nature Biotechnology 14: 845-51 (1996). :826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13 :65-93 (1995).

Finally, human antibodies can also be generated in vitro by activated B cells (see, US Pat. Nos. 5,567,610 and 5,229,275).

5) Antibody fragments

In some cases, it is advantageous to use antibody fragments rather than whole antibodies. The smaller fragment size allows for rapid clearance and results in improved engagement with solid tumors.

A variety of techniques have been developed for producing antibody fragments. Traditionally, these fragments were derived by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., J Biochem Biophys. Method. 24 : 107-117 (1992); and Brennan et al., Science 229 : 81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing for the easy production of large quantities of these fragments. Antibody fragments can be isolated from the phage antibody libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab') ₂ fragments (Carter et al., Bio/Technology 10 : 163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell cultures. ₂ fragments. Fab and F(ab') ₂ with increased in vivo half-life are described in U.S. Patent No. 5,869,046. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv). See WO93/16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458. Antibody fragments can also be "linear antibodies," for example, as described in U.S. Patent No. 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

6) Antibody-dependent enzyme-mediated prodrug therapy (ADEPT)

The antibodies of the invention can also be used in ADEPT by coupling the antibodies to a prodrug-activating enzyme that converts prodrugs (e.g., peptidyl chemotherapeutic agents, see WO 81/01145) into active anticancer drugs. See, e.g., WO 88/07378 and U.S. Pat. No. 4,975,278.

The enzyme component of the immunoconjugates useful in ADEPT includes any enzyme capable of acting on a prodrug to convert it into its more active, cytotoxic form.

Enzymes that can be used in the methods of the present invention include, but are not limited to, glycosidases, glucose oxidase, human lysozyme, human glucuronidase, alkaline phosphatase, for converting phosphate-containing prodrugs into free drugs; arylsulfatase, for converting sulfate-containing prodrugs into free drugs; cytosine deaminase, for converting non-toxic 5-fluorocytosine into the anticancer drug 5-fluorouracil; proteases, such as Serratia protease, thermolysin, subtilisin, carboxypeptidases (e.g., carboxypeptidase G2 and carboxypeptidase A), and cathepsins (e.g., tissue proteases). [0014] Examples of the present invention include prodrugs comprising prodrugs of the invention, such as proteases B and L (proteinases B and L), for converting peptide-containing prodrugs into free drugs; D-alanamide carboxypeptidase, for converting prodrugs containing D-amino acid substitutes; carbohydrate-cleaving enzymes, such as β-galactosidase and neuraminidase, for converting glycosylated prodrugs into free drugs; β-lactamase, for converting β-lactam-derived drugs into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, for converting drugs derived from their ammonia nitrogen by phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity (also referred to in the art as "abzymes") can be used to convert the prodrugs of the invention into free active drugs (see, e.g., Massey, Nature 328 : 457-458 (1987)). Antibody-abzyme conjugates can be prepared as described herein to deliver the abzyme to tumor cell populations.

The enzymes can be covalently bound to the polypeptides or antibodies described herein using techniques known in the art, such as the heterobifunctional cross-linking agents described above. Alternatively, recombinant DNA techniques familiar to those skilled in the art can be used to construct fusion proteins comprising at least the antigen-binding region of an antibody herein linked to at least one functionally active portion of an enzyme of the invention (see, e.g., Neuberger et al., Nature 312 : 604-608 (1984)).

7) Bispecific and multispecific antibodies

Bispecific antibodies (BsAbs) are antibodies that have binding specificity for at least two different epitopes (including those on the same protein or on another protein). Alternatively, one arm can bind to the target antigen, and the other arm can be combined with an arm that binds to a triggering molecule on a leukocyte, thereby concentrating and confining the cellular defense mechanism to the target antigen-expressing cell, such as a T cell receptor molecule (e.g., CD3), or an IgG Fc receptor (FcγR) such as FcγR1 (CD64), FcγRII (CD32), and FcγRIII (CD16). Such antibodies can be derived from full-length antibodies or antibody fragments (e.g., F(ab') ₂ bispecific antibodies).

Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing target antigens. Such antibodies have one arm that binds to the desired antigen and another arm that binds to the cytotoxic agent, such as saporin, anti-interferon-α, vinca alkaloids, ricin A chain, methotrexate, or a radioactive isotope hapten. Examples of known bispecific antibodies include anti-ErbB2/anti-FcgRIII (WO 96/16673), anti-ErbB2/anti-FcgRI (U.S. Patent No. 5,837,234), and anti-ErbB2/anti-CD3 (U.S. Patent No. 5,821,337).

Methods for generating bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two pairs of immunoglobulin heavy chains/light chains, wherein the two chains have different specificities. Millstein et al., Nature 305 : 537-539 (1983). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) generate a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, typically by affinity chromatography steps, is quite cumbersome and the product yield is low. Similar methods are disclosed in WO 93/08829 and Traunecker et al., EMBO J. 10 : 3655-3659 (1991).

According to different methods, the antibody variable domain with the desired binding specificity (antibody-antigen binding site) is fused with the immunoglobulin constant domain sequence. Preferably, it is fused with the immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2 and CH3 regions. Preferably, the first heavy chain constant region (CH1) comprising the site necessary for light chain binding is present in at least one fusion. The DNA encoding the immunoglobulin heavy chain fusion and the immunoglobulin light chain when needed are inserted into separate expression vectors and co-transfected into a suitable host organism. In embodiments where the optimal yield is provided when the three polypeptide chains used to construct are unequal in ratio, this provides great flexibility for adjusting the mutual ratio of the three polypeptide fragments. However, when at least two polypeptide chains are expressed in the same ratio resulting in high yield or when the ratio has no particular significance, it is possible to insert the coding sequence of two or all three polypeptide chains into one expression vector.

In a preferred embodiment of the method, bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity on one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) on the other arm. Since the presence of immunoglobulin light chain only in half a bispecific molecule provides a convenient separation approach, it is found that this asymmetric structure is convenient for separating the desired bispecific compound from the unwanted immunoglobulin chain combination. The method is disclosed in WO94/04690. For further details about generating bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology 121 : 210 (1986).

According to another method of recording in WO96/27011 or United States Patent (USP) 5,731,168, the interface between a pair of antibody molecules can be transformed to maximize the percentage of the heterodimer that will be recovered from recombinant cell culture.Preferred interface comprises at least part of the CH3 district of antibody constant domain.In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with larger side chains (for example tyrosine or tryptophan).By replacing with smaller amino acid side chains (for example alanine or threonine) of large amino acid side chains, on the interface of the second antibody molecule, produce compensatory " cavity " that is identical or similar with the large side chain size.This provides the mechanism that improves heterodimer output such as homodimer than other undesirable end products.

The technology of generating bispecific antibodies from antibody fragments has been documented in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229 :81 (1985) described a method for generating F(ab') ₂ fragments by proteolytic cleavage of intact antibodies. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and prevent the formation of intermolecular disulfide bonds. The resulting Fab' fragments are then converted into thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconstituted into a Fab'-TNB derivative to form a bispecific antibody. The resulting bispecific antibodies can be used as selective immobilization reagents for enzymes.

Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175 : 217-225 (1992) described the generation of fully humanized bispecific antibody F(ab') ₂ molecules. Each Fab' fragment was secreted separately from E. coli and subjected to directed chemical coupling in vitro to form bispecific antibodies. The bispecific antibodies thus formed were able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

A variety of techniques for directly generating and isolating bivalent antibody fragments from recombinant cell culture have also been described. For example, leucine zippers have been used to generate bivalent heterodimers. Kostelny et al., J. Immunol. 148 (5): 1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90 : 6444-6448 (1993) provides an alternative mechanism for generating bispecific/bivalent antibody fragments. The fragments comprise a heavy chain variable domain ( _VH ) and a light chain variable domain ( _VL ) connected by a linker that is too short to allow pairing between the two domains on the same chain. Thus, the _VH and _VL domains of one fragment are forced to pair with the complementary _VL and _VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for generating bispecific/bivalent antibody fragments using single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152 : 5368 (1994).

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147 : 60 (1991).

Exemplary bispecific antibodies can bind to two different epitopes on a given molecule. Alternatively, the anti-protein arm can be combined with an arm that binds to a triggering molecule on a leukocyte, thereby focusing the cellular defense mechanism on cells expressing the specific protein, such as T cell receptor molecules (e.g., CD2, CD3, CD28 or B7), or IgG Fc receptors (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). Bispecific antibodies can also be used to confine cytotoxic agents to cells expressing specific proteins. Such antibodies have a protein binding arm and an arm that binds a cytotoxic agent or a radionuclide chelator (such as EOTUBE, DPTA, DOTA or TETA). Another bispecific antibody of interest binds to a protein of interest and further binds to tissue factor (TF).

8) Multivalent Antibodies

Multivalent antibodies can be internalized (and/or catabolized) by cells expressing the antigen to which the antibody binds faster than bivalent antibodies. The antibodies of the present invention can be multivalent antibodies (other than the IgM class) having three or more antigen binding sites (e.g., tetravalent antibodies) that can be easily generated by recombinant expression of nucleic acids encoding antibody polypeptide chains. Multivalent antibodies may comprise a dimerization domain and three or more antigen binding sites. Preferred dimerization domains comprise (or consist of) an Fc region or a hinge region. In this case, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. Preferred multivalent antibodies herein comprise (or consist of) three to about eight, but preferably four, antigen binding sites. Multivalent antibodies comprise at least one polypeptide chain (preferably two polypeptide chains), wherein the polypeptide chain comprises two or more variable domains. For example, the polypeptide chain may comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is the first variable domain, VD2 is the second variable domain, Fc is a polypeptide chain of the Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, the polypeptide chain may comprise: VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (preferably four) light chain variable domain polypeptides. The multivalent antibody herein may comprise, for example, about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides encompassed herein comprise a light chain variable domain and optionally further comprise a CL domain.

9) Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies consist of two covalently linked antibodies. For example, one antibody in the heteroconjugate can be linked to avidin and the other to biotin. For example, such antibodies have been proposed for targeting immune system cells to unwanted cells (U.S. Patent No. 4,676,980) and for treating HIV infection (WO 91/00360, WO 92/200373, and EP 0308936). It is contemplated that the antibodies can also be prepared in vitro using known synthetic protein chemistry methods, including those utilizing cross-linking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate, and those disclosed, for example, in U.S. Patent No. 4,676,980. Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in US Pat. No. 4,676,980, along with a number of cross-linking techniques.

10) Effector function engineering

It may be desirable to modify the antibodies of the invention with respect to Fc effector function, for example, to modify (e.g., enhance or eliminate) the antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) of the antibody. In a preferred embodiment, the Fc effector function of the anti-PD-L1 antibody is reduced or eliminated. This can be accomplished by introducing one or more amino acid substitutions into the Fc region of the antibody. Alternatively or additionally, cysteine residues may be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp. Med 176: 1191-1195 (1992) and Shopes, B., J. Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53:2560-2565 (1993). Alternatively, antibodies can be engineered to have dual Fc regions, thereby enhancing complement lysis and ADCC capabilities. See Stevenson et al., Anti - Cancer Drug Design 3:219-230 (1989).

To extend the serum half-life of an antibody, a salvage receptor binding epitope can be incorporated into the antibody (particularly an antibody fragment) as described, for example, in U.S. Patent No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG ₁ , IgG ₂ , IgG ₃ or IgG ₄ ) that is responsible for extending the serum half-life of the IgG molecule in vivo.

11) Other amino acid sequence modifications

Amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing suitable nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions and/or insertions of residues and/or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be performed to obtain the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes may also alter post-translational processes of the antibody, such as altering the number or location of glycosylation sites.

A useful method for identifying certain residues or regions of antibodies that are preferred positions for mutation is called "alanine partitioning mutagenesis," as described by Cunningham and Wells in Science, 244 : 1081-1085 (1989). Here, one or a group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to influence the interaction of the amino acid antigen. The amino acid positions that are then selected to show functional sensitivity to the replacement are then introduced at or about the replacement site. Therefore, when the site for introducing an amino acid sequence change is predetermined, the nature of the mutation itself does not need to be predetermined. For example, in order to analyze the performance of a mutation at a given site, alanine partitioning mutagenesis or random mutagenesis is performed at the target codon or target region and the antibody variants expressed are screened for activity as needed.

Amino acid sequence insertions include amino and/or carboxyl terminal fusions ranging in length from one residue to polypeptides comprising more than one hundred residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to a cytotoxic polypeptide. Other insertion variants of the antibody molecule include fusion of the N-terminus or C-terminus of the antibody with an enzyme (e.g., with respect to ADEPT) or a polypeptide that increases the serum half-life of the antibody.

The variant of another type is amino acid substitution variant.These variants have at least one amino acid residue in the antibody molecule replaced by different residues.The most interesting site that is used for substitution mutation occurs comprises hypervariable region, but also considers that FR changes.Conservative substitutions are shown in Table A below under the title of "preferred substitution".If this type of substitution causes the variation of biological activity, then more substantial changes are presented in the "exemplary substitutions" in Table A, or further described below with reference to amino acid class, can be introduced and screen product.

Table A

Amino acid substitution

Starting residue Exemplary substitutions Preferred substitutions Ala(A) val;leu;ile val Arg(R) lys;gln;asn lys Asn(N) gln;his;asp,lys;arg gln Asp(D) glu;asn glu Cys(C) ser;ala ser Gln(Q) asn;glu asn Glu(E) asp;gln asp Gly(G) ala ala His(H) asn;gln;lys;arg arg Ile(I) leu; val; met; ala; phe; norleucine leu Leu(L) norleucine;ile;val;met;ala;phe ile Lys(K) arg;gln;asn arg Met(M) leu; phe; ile leu Phe(F) leu;val;ile;ala;tyr tyr Pro(P) Ala ala Ser(S) Thr thr Thr(T) Ser ser Trp(W) tyr; phe tyr Tyr(Y) trp;phe;thr;ser phe Val(V) ile; leu; met; phe; ala; norleucine leu

Substantial modifications in the biological properties of the antibody can be made by selecting substitutions that differ significantly in their effect on maintaining: (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are grouped into groups based on common side chain properties:

(1) Hydrophobic: norleucine, met, ala, val, leu, ile;

(2) Neutral hydrophilicity: cys, ser, thr;

(3) Acidic: asp, glu;

(4) Basic: asn, gln, his, lys, arg;

(5) Residues that affect chain orientation: gly, pro; and

(6) Aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another.

Any cysteine residue not involved in maintaining the proper conformation of the antibody may also be substituted with serine to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine bonds may be added to the antibody to improve its stability (particularly when the antibody is an antibody fragment such as an Fv fragment).

Particularly preferred types of substitution variants include one or more hypervariable region residues that replace the parent antibody (e.g., humanized or human antibody). Generally, the variants obtained with respect to further development have improved biological properties relative to the parent antibody from which they are produced. A convenient way to produce such substitution variants involves affinity maturation using phage display. In short, some hypervariable region sites (e.g., 6-7 sites) are mutated to produce all possible amino substitutions at each site. The antibody variants thus produced are displayed in a monovalent manner from filamentous phage particles as fused to the gene III product of M13 packaged in each particle. The phage display variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or additionally, analyzing the crystal structure of the antigen-antibody complex to identify the contact points between the antibody and its target (e.g., PD-L1, B7.1) can be beneficial. Such contact residues and neighboring residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, the panel of variants is screened as described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

Another type of amino acid variant of an antibody alters the original glycosylation pattern of the antibody. By altering, one or more carbohydrate moieties found in the antibody are deleted, and/or one or more glycosylation sites are added that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

Typically, glycosylation sites are added to the antibody by altering the amino acid sequence so that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Changes can also be made by adding or substituting one or more serine or threonine residues into the starting antibody sequence (for O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of the antibodies of the invention are prepared by a variety of methods known in the art, including, but not limited to, isolation from natural sources (in the case of naturally occurring amino acid variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or non-variant form.

12) Other Antibody Modifications

The antibodies of the present invention can be further modified to include other non-proteinaceous structural parts known in the art and easily obtained. Preferably, the structural part suitable for the derivatization of the antibody is a water-soluble polymer. Non-limiting examples of water-soluble polymers include, but are not limited to: polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyols (such as glycerol), polyvinyl alcohol and mixtures thereof. Due to its stability in water, polyethylene glycol propionaldehyde can have advantages in production. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers connected to the antibody can vary, and if more than one polymer is connected, they can be the same or different molecules. Generally, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc. Such techniques and other appropriate formulations are disclosed in Remington: The Science and Practice of Pharmacy, 20th Ed., Alfonso Gennaro, ed., Philadelphia College of Pharmacy and Science (2000).

D. Pharmaceutical preparations

The therapeutic agent can be prepared for storage by mixing the active ingredient having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington: The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wiklins, Pub., Gennaro Ed., Philadelphia, PA 2000). Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers, antioxidants including ascorbic acid, methionine, vitamin E, sodium metabisulfite; preservatives, isotonic agents, stabilizers, metal complexes (e.g., Zn-protein complexes); chelating agents such as EDTA and/or nonionic surfactants.

When the therapeutic agent is an antibody fragment, it is preferably a minimal inhibitory fragment that specifically binds to the binding domain of the target protein. For example, based on the variable region sequence of the antibody, such antibody fragments or even peptide molecules can be designed that retain the ability to bind to the target protein sequence. The peptides can be produced by chemical synthesis and/or recombinant DNA technology. (See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90 : 7889-7893 [1993]).

Buffers are used to control the pH within a range that optimizes therapeutic efficacy, particularly when stability is pH-dependent. Preferred concentrations of buffers range from about 50 mM to about 250 mM. Suitable buffers for use in the present invention include organic and inorganic acids, and their salts. For example, citrates, phosphates, succinates, tartrates, fumarates, gluconates, oxalates, lactates, and acetates. Additionally, buffers can be composed of histidine and trimethylamine salts, such as Tris.

Preservatives are added to inhibit microbial growth, typically in the range of 0.2% to 1.0% (w/v). Suitable preservatives for use in the present invention include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

Tonicity agents, sometimes referred to as "stabilizers," are included to adjust or maintain the liquid tonicity of the composition. When large, charged biomolecules such as proteins and antibodies are used, they are often referred to as "stabilizers" because they can interact with the charged groups of amino acid side chains, thereby reducing the possibility of intra- and intermolecular interactions. Taking into account the relative amounts of the other ingredients, the tonicity agent may be present in any amount between 0.1% and 25%, preferably between 1 and 5%, by weight. Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol.

Additional excipients include agents that can act as one or more of the following: (1) fillers, (2) solubility enhancers, (3) stabilizers, and (4) agents that inhibit denaturation or adhesion to the container walls. Such excipients include: polyhydroxy sugar alcohols (listed above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, and the like; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclopolyols, t-Butyl succinate ... Alcohols (e.g., cyclohexanehexol), polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose; disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

A nonionic surfactant or detergent (also known as a "wetting agent") is included to aid in the dissolution of the therapeutic agent and to protect the therapeutic protein from aggregation caused by agitation. It also allows the formulation to be subjected to shear surface stress without causing denaturation of the active therapeutic protein or antibody. The nonionic surfactant is present in the range of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07 mg/ml to about 0.2 mg/ml.

Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), polyols, polyoxyethylene sorbitan monoethers (-20, -80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glyceryl monostearate, fatty acid sugar esters, methylcellulose and carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyl sodium sulfonate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

In order to use the formulations for in vivo administration, they must be sterile. The formulations can be sterilized by filtration through a sterile filter membrane. The therapeutic compositions herein are generally placed in a container with a sterile access port, for example, an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle.

The route of administration is according to known and recognized methods, for example by single or multiple bolus administration, or by infusion over a prolonged period of time in a suitable manner, for example, by injection or infusion by the subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular route, by topical application, by inhalation or by sustained release or delayed release methods.

The formulations herein may also contain more than one active compound, as necessary for the specific condition being treated, preferably compounds having complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may include a cytotoxic agent, cytokine, or growth inhibitory agent. Such molecules are suitably present in combination in amounts effective for their intended purpose.

The active ingredient may also be embedded in microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, prepared, for example, by coascervation techniques or interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules, and poly-(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 18th edition (supra).

The stability of the proteins and antibodies described herein can be enhanced by the use of non-toxic "water-soluble polyvalent metal salts". Examples include Ca2 ⁺ , Mg2 ⁺ , Zn2 ⁺ , Fe2 ⁺ , Fe3 ⁺ , Cu2 ⁺ , Sn2 ⁺ , Sn4 ⁺ , Al2 ⁺ and Al3 ⁺ . Examples of anions that can form water-soluble salts with the above-mentioned polyvalent metal cations include those formed from inorganic acids and/or organic acids. Such water-soluble salts have a solubility in water (20°C) of at least about 20 mg/ml, alternatively at least about 100 mg/ml, alternatively at least about 200 mg/ml.

Suitable inorganic acids that can be used to form the "water-soluble polyvalent metal salt" include hydrochloric acid, acetic acid, sulfuric acid, nitric acid, thiocyanic acid and phosphoric acid. Suitable organic acids that can be used include aliphatic carboxylic acids and aromatic acids. Aliphatic acids under this definition can be defined as saturated or unsaturated _C2-9 carboxylic acids (e.g., aliphatic mono-, di- and tricarboxylic acids). For example, exemplary monocarboxylic acids under this definition include saturated _C2-9 monocarboxylic acids, acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid and capryonic acid, and unsaturated _C2-9 monocarboxylic acids, acrylic acid, propriolic methacrylic acid, crotonic acid and isocrotonic acid. Exemplary dicarboxylic acids include saturated _C2-9 dicarboxylic acids, malonic acid, succinic acid, glutaric acid, adipic acid and pimelic acid, while unsaturated _C2-9 dicarboxylic acids include maleic acid, fumaric acid, citraconic acid and methylfumaric acid. Exemplary tricarboxylic acids include the saturated _C2-9 tricarboxylic acids, tricarballylic acid, and 1,2,3-butanetricarboxylic acid. Additionally, carboxylic acids within this definition may also contain one or two hydroxyl groups to form hydroxycarboxylic acids. Exemplary hydroxycarboxylic acids include glycolic acid, lactic acid, glyceric acid, tartaric acid, malic acid, tartaric acid, and citric acid. Aromatic acids within this definition include benzoic acid and salicylic acid.

Commonly used water-soluble multivalent metal salts that can be used to help stabilize the encapsulated polypeptides of the present invention include, for example: (1) halogenated inorganic acid metal salts (e.g., zinc chloride, calcium chloride), sulfates, nitrates, phosphates, and thiocyanates; (2) aliphatic carboxylic acid metal salts (e.g., calcium acetate, zinc acetate, calcium propionate, zinc glycolate, calcium lactate, zinc lactate, and zinc tartrate); and (3) aromatic carboxylic acid metal salts, benzoates (e.g., zinc benzoate), and salicylates.

E. Treatment methods

For the prevention or treatment of disease, the appropriate dosage of the active agent will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the agent is administered for preventive or therapeutic purposes, previous treatment, the patient's medical history and response to the agent, and the judgment of the attending physician. The agent is suitably administered to the patient at one time or over a series of treatments.

In a specific embodiment, the invention relates to attenuation of co-stimulation resulting from signaling through PD-1 (particularly by the use of PD-L1 antibodies that block binding of PD-1 and/or B7.1), and therapeutic treatment of T cell dysfunction disorders.

1. Infection

PD-1 and its ligand ("PD-1:PD-L") play an important role in regulating immune defense against pathogens that cause acute and chronic infections. PD-1:PD-L signaling plays a key role in regulating the balance between effective antimicrobial immune defense and immune-mediated tissue damage. For example, although PD-1 knockout mice clear adenovirus infection faster than their wild-type counterparts, they develop more severe hepatocellular damage. Iwai et al., J. Exp. Med. (Journal of Experimental Medicine) 198 : 39-50 (2003). In a mouse model of herpes simplex virus stromal keratitis, blocking anti-PD-L1 antibodies aggravated keratitis and increased HSV-1-specific effector CD4 T cell expansion and IFN-γ production and survival. Jun et al., FEBS Lett. 579 : 6259-64 (2005).

Microorganisms that cause chronic infection use the PD-1:PD-L signaling pathway to evade the host immune response, leading to chronic infection. Viruses that cause chronic infection can render virus-specific T cells nonfunctional and thereby silence antiviral T cell responses. Barber et al., Nature 439 : 682-87 (2006); Wherry et al., J. Virol. 78 : 5535-45 (2004). T cell exhaustion or anergy of CD8 ⁺ T cells is an important reason for the ineffective control of viruses during chronic infection and is a characteristic of chronic LCMV infection in mice, HIV, HBV, HCV and HTLV infection in humans, and SIV infection in primates. The exhausted virus-specific CD8 ⁺ T cell phenotype appears to have a graded, progressive loss of function, with IL-2 production and cytotoxicity lost first, followed by the production of effector cytokines.

In mice with chronic LCMV infection, PD-1 is upregulated upon activation and maintained at high levels by exhausted CD8 ⁺ T cells. Barber et al., supra. Administration of an antibody that blocks PD-1:PD-L1 binding leads to enhanced T cell responses and a substantial reduction in viral load. In mice with persistent infection accompanied by an ineffective CD4 ⁺ _TH response, blocking PD-1:PD-L1 restored CD8 ⁺ T cells from a dysfunctional state, resulting in proliferation, cytokine secretion, killing of infected cells, and reduced viral load, strongly suggesting a therapeutic approach for treating chronic viral infection.

Because of the role of PD-1:PD-L in LCMV, there has been strong interest in targeting this pathway to treat chronic infection in humans. PD-1 is highly expressed on HIV-specific T cells [Petrovas et al., J. Exp. Med. 203 :2281-92 (2006); Day et al., Nature 443 :350-54 (2006); Traumann et al., Nat. Med. 12 :1198-202 (2006)], HBV-specific T cells [Boettler et al., J. Virol. 80 :3532-40 (2006); Boni et al., J. Virol. 81 :4215-25 (2007)], and HCV-specific T cells [Urbani et al., J. Virol. 80 :11398-403 (2006)]. PD-L1 is also upregulated on peripheral blood CD14 ⁺ monocytes and bone marrow DCs in patients with chronic HBV infection [Chen et al., J. Immunol. 178 : 6634-41 (2007); Geng et al., J. Viral Hepat. 13 : 725-33 (2006)], and on CD14+ cells and T cells in HIV patients [Trabattoni et al., Blood 101 : 2514-20 (2003)]. In vitro blocking of the PD-1: PD-L1 interaction reverses the exhaustion of HIV-specific, HBV-specific, HCV-specific, and SIV-specific CD8+ and CD4+ T cells and restores proliferation and cytokine production. Petrovas et al., J. Exp. Med. 203:2281-92 (2006); Day et al., supra; Trautmann et al., supra; Boni et al., supra; Urbani et al., supra; Velu et al., J. Virol. 81 :5819-28 (2007).

The expression level of PD-1 can also be a useful diagnostic marker on virus-specific CD8 ⁺ T cells, used to indicate the degree of T cell exhaustion and disease severity. The expression level of PD-1 on HIV-specific CD8 ⁺ T cells is correlated with viral load, reducing CD ⁴ + counts and reducing the ability of CD8 ⁺ T cells to proliferate in response to HIV antigens in vitro. Corresponding to in vivo observations, there is a direct correlation between the expression of PD-1 on HIV-specific CD4 ⁺ T cells and viral load. D'Souza et al., J. Immunol. (Journal of Immunology) 179 : 1979-87 (2007). Long-term non-progressors have functional HIV-specific memory CD8 ⁺ T cells with significantly lower PD-1 expression, which is in contrast to typical progressors, who express significantly upregulated PD-1, which is associated with reduced CD4 ⁺ T cell numbers, reduced CD4 ⁺ T cell numbers, reduced HIV-specific effector memory CD8 ⁺ T cell function and increased plasma viral load. Zhang et al., Blood 109 :4671-78 (2007).

The PD-1:PD-L pathway is also involved in the chronicity of bacterial infection. Helicobacter pylori causes chronic gastritis and gastroduodenal ulcers and is a risk factor for gastric cancer. During H. pylori infection, T cell responses are insufficient to clear the infection, leading to persistent infection. PD-L1 is upregulated on gastric epithelial cells after exposure to H. pylori in vitro or in vivo. Gastric epithelial cells express MHC class II molecules and are thought to play an important APC function during H. pylori infection. Anti-PD-L1 antibodies that block the interaction between PD-1 and PD-L1 enhance T cell proliferation and IL-2 production in gastric epithelial cell cultures exposed to H. pylori and in CD4 T cells. Blocking PD-L1 with antibodies or siRNA prevents the generation of regulatory T cells, suggesting that PD-L1 can promote T cell suppression and persistent infection by controlling the dynamics between regulatory and effector T cells during H. pylori infection. Beswick et al., Infect. Immun. 75 : 4334-41 (2007).

Parasites also use the PD-1:PD-L1 pathway to induce macrophages that suppress immune responses. During infection with Taenia crassiceps (i.e., tapeworms), PD-1 and PD-L2 are upregulated on activated macrophages, and CD4+ T cells express PD-1. Blocking PD-1, PD-L1 or PD-L2 significantly reduces the inhibition of in vitro T cell proliferation by macrophages (which come from mice infected with tapeworms). Terrazas et al., Int. J. Parasitol. 35 : 1349-58 (2005). During infection with Schistosoma mansoni in mice, macrophages express high levels of PD-L1 and more moderate levels of PD-L2. Anti-PD-L1 removes the ability of these macrophages to suppress T cell proliferation in vitro, while anti-PD-L2 has no effect. PD-L1 expression on macrophages from infected mice decreased 12 weeks after infection and correlated with the disruption of T cell anergy. Smith et al., J. Immunol. 173 : 1240-48 (2004).

2. Tumor Immunity

Experimental evidence for tumor immunity includes (i) observations of spontaneous remissions, (ii) the presence of detectable but ineffective host immune responses to tumors, (iii) an increased prevalence of primary and secondary malignancies in immunocompromised patients, (iv) increased levels of antibodies and T lymphocytes detected in patients with tumors, and (v) the observation that test animals can be immunized against a variety of tumor types.

Studies have shown that most human tumors express tumor-associated antigens (TAAs), which can be recognized by T cells and therefore may induce an immune response. Boon et al., Immunol. Today 16 : 334-336 (1995). Early clinical trials have been initiated that vaccinate cancer patients with TAAs or pulse professional antigen-presenting cells with TAAs. Dudley et al., Science 298 : 850-854 (2002); Gajewski et al., Clin. Cancer Res. 7 : 895s-901s (2001); Marincola et al., Adv. Immunol. 74 : 181-273 (2000); Peterson et al., J. Clin. Oncol. 21 : 2342-2348 (2003). Induction of tumor antigen-specific CD8+ T cells has been achieved in many of these trials. Mackensen et al., Eur. Cytokine Netw 10 :329-336 (1999); Peterson et al., supra. Adoptive transfer of tumor antigen-specific T cells into patients has also been performed, and targeting of expanded cytotoxic T lymphocytes (CTLs) to tumor sites has been demonstrated. Meidenbauer et al., J. Immunol. 170 :2161-2169 (2003). However, despite tumor infiltration of immune effector cells, tumor growth is rarely controlled.

It is well established that the tumor microenvironment can protect tumor cells from immune destruction. Ganss et al., Cancer Res. 58 : 4673-4681 (1998); Singh et al., J. Exp. Med. 175 : 139-146 (1992). Soluble factors and membrane-bound molecules including transforming growth factor β (TGF-β), interleukin (IL)-10, prostaglandin E ₂ , FASL, CTLA-4 ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and programmed death receptor ligand 1 (PD-L1, aka B7-H1) have been found to be expressed by tumors and are believed to mediate immune evasion. Therefore, blocking these negative immunoregulatory signals on tumor cells is a promising approach to enhance tumor-specific CD8+ T cell immunity in vivo.

PD-L1 expression on many tumors is a component of this inhibition and may synergize with other immunosuppressive signals. PD-L1 negatively regulates T cell receptor signaling. PD-L1 expression has been shown in situ in a wide variety of solid tumors, including breast, lung, colon, ovarian, melanoma, bladder, liver, salivary gland, gastric, glioma, thyroid, thymic, epithelial, and head and neck cancers. Brown et al., J. Immunol. 170 :1257-66 (2003); Dong et al., Nat. Med. 8 :793-800 (2002); Hamanishi et al., PNAS 104 :3360-65 (2007); Strome et al., Cancer Res. 63 :6501-5 (2003); Inman et al., Cancer 109 :1499-505 (2007); Konishi et al., Clin. Cancer Res. 10 :5094-100 (2004); Nakanishi et al., Cancer Immunol. Immunother. 56 :1173-82 (2007); Nomi et al., Clin. Cancer Res. 13 :2151-57 (2004); Thompson et al., PNAS 101 :17174-79 (2004); Wu et al., Acta Histochem. 108 :19-24 (2006).

Immunostaining also revealed the expression of PD-1:PD-L in various cancers.

Interestingly, cancer has been characterized as a chronic inflammatory disease. Coussens et al., Nature 420 :860-867 (2002). Although up to 15% of cancers worldwide have a direct infectious etiology [Kuper et al., J. Intern. Med. 248 :171-183 (2000)], many human tumors are associated with chronic irritation and inflammation. Zou et al., Ntu. Rev. Cancer 5 :263-274 (2005).

Studies correlating PD-L1 expression on tumors with disease outcome have shown that PD-L1 expression is strongly associated with poor prognosis in renal, ovarian, bladder, breast, gastric, and pancreatic cancers, but may not be associated with non-small cell lung cancer. Hamanishi et al., Proc. Natl. Acad. Sci. USA 104 : 3360-65 (2007), Inman et al., Cancer 109 : 1499-505 (2007), Konishi et al., Clin. Cancer Res. 10 : 5094-100 (2004); Nakanishi et al., Cancer Immunol. Immunother. 56 : 1173-82 (2007); Nomi et al., Clin. Cancer Res. 13 : 2151-57 (2007); Thompson et al., Proc. Natl. Acad. Sci. USA 101 : 17174-79 (2004); Wu et al., Acta Histochem. 108 :19-24(2006). In addition, these studies have shown that higher levels of PD-L1 expression on tumors can promote tumor stage progression and invasion into deeper tissue structures.

The PD-1:PD-L pathway may also play a role in hematological malignancies. PD-1 or PD-L1 are rarely expressed in B-cell malignancies, but PD-L2 is overexpressed in mantle cell malignancies. Brown et al., supra; Rosenwald et al., J. Exp. Med. (Journal of Experimental Medicine) 198 : 851-62 (2003). PD-L1 is expressed on multiple myeloma cells, but not on normal plasma cells. The expansion of T cell responses to myeloma cells is enhanced in vitro by PD-L1 blockade. Liu et al., Blood (Blood) 110 : 296-304 (2007). PD-L1 is expressed on some primary T-cell lymphomas, particularly anaplastic large cell T lymphoma, and PD-L1 is expressed on the associated follicular dendritic cell network. Dorfman et al., Am. J. Surg. Pathol. 30 : 802-10 (2006). Microarray analysis further showed that tumor-associated T cells responded to in situ PD-1 signals in Hodgkin's lymphoma. Chemnitz et al., Blood 110 : 3226-33 (2007). PD-1 and PD-L1 are expressed on CD4 ⁺ T cells in HTLV-1-mediated adult T-cell leukemia and lymphoma. Shimauchi et al., Int. J. Cancer 121 : 2585-90 (2007). These tumor cells are hyporesponsive to TCR signals, and PD-1 blockade increases their expression of TNF-α, but does not increase IFN-γ. Studies in animal models have demonstrated that PD-L1 expression on tumors inhibits T cell activation and tumor cell lysis, leading to increased tumor-specific T cell death in some cases. Dong et al., Nat. Med. 8 :793-800 (2006); Hirano et al., Cancer Res. 65 :1089-96 (2005).

Therefore, the anti-PD-L1 antibodies of the present invention inhibit signaling through PD-L1, thereby enhancing T cell function and showing promise in reducing tumor immunity, thereby being effective in treating cancer.

F. Combination Therapy

The methods of the present invention may be combined with known methods for treating chronic infections or cancer, either as a combined or additional treatment step or as an additional component of a therapeutic formulation.

1. Cancer :

Enhancing the host's immune function to resist tumors is an increasingly interesting topic. Conventional methods include (i) APC enhancement, such as (a) injecting DNA encoding heterologous MHC alloantigens into the tumor, or (b) transfecting biopsied tumor cells with genes that increase the possibility of immune antigen recognition (e.g., immunostimulatory cytokines, GM-CSF, costimulatory molecules B7.1, B7.2), (iii) adoptive cellular immunotherapy, or treatment with activated tumor-specific T cells. Adoptive cellular immunotherapy includes separating tumor-infiltrating host T-lymphocytes, such as by IL-2 or tumor or both stimulating the in vitro expansion of the population. Additionally, the isolated T cells of dysfunctional disorders can also be activated by the anti-PD-L1 antibodies of the present invention applied in vitro. The T cells activated in this way can then be re-administered to the host.

Conventional cancer treatments include the following: (i) radiation therapy (e.g., radiotherapy, X-ray therapy, irradiation), or the use of ionizing radiation to kill cancer cells and shrink tumors. Radiation therapy can be administered via external beam radiotherapy (EBRT) or internal brachytherapy; (ii) chemotherapy, or the use of cytotoxic drugs, which generally affect rapidly dividing cells; (iii) targeted therapy, or agents that specifically affect the deregulation of cancer cell proteins (e.g., the tyrosine kinase inhibitors imatinib, gefitinib; monoclonal antibodies, photodynamic therapy); (iv) immunotherapy, or enhancing the host immune response (e.g., vaccines); (v) hormone therapy, or blocking hormones (e.g., when the tumor is hormone-sensitive); (vi) angiogenesis inhibitors, or blocking the formation and growth of blood vessels; and (vii) palliative care, or treatments that involve improving the quality of care to reduce pain, nausea, vomiting, diarrhea, and bleeding. Pain medications such as morphine and oxycodone, and anti-emetics such as ondansetron and aprepitant, may allow for more aggressive treatment regimens.

In the treatment of cancer, any of the above conventional cancer immunotherapy treatments can be administered before, after, or simultaneously with the administration of the anti-PD-L1 antibodies of the present invention. In addition, the anti-PD-L1 antibodies of the present invention can be administered before, after, or simultaneously with conventional cancer treatments, such as administration of tumor-binding antibodies (e.g., monoclonal antibodies, toxin-conjugated monoclonal antibodies) and/or administration of chemotherapeutic agents.

2. Infection :

In treating infections (e.g., acute and/or chronic infections), administration of the anti-PD-L1 antibodies of the present invention may be used in addition to or in place of conventional treatments that stimulate natural host immune defenses against infection. Natural host immune defenses against infection include, but are not limited to, inflammation, fever, antibody-mediated host defenses, T lymphocyte-mediated host defenses, including lymphokine secretion and cytotoxic T cells (particularly during viral infections), complement-mediated lysis and opsonization (promoting phagocytosis), and phagocytosis. The ability of the anti-PD-L1 antibodies of the present invention to revitalize dysfunctional T cells will be particularly useful in treating chronic infections, particularly those in which cell-mediated immunity is critical for full recovery.

a. Bacteria

For infections caused by bacterial infections, the anti-PD-L1 antibodies of the present invention can be administered concurrently with, before, or after standard therapies for treating bacterial infections. Bacterial infections are most commonly treated with antibacterial antibiotics, but serum containing pathogen-specific antibodies from immunized hosts is also effective.

For bacteria that cause disease by secreting toxins (toxigenic bacteria), vaccination with inactivated toxins and/or administration of therapeutic agents that block the toxicity of the toxins are often effective (e.g., polyclonal sera, antibodies, antibiotics, etc.). These organisms include Clostridium spp., Bacillus spp., Corynebacterium spp., Vibrio chloerae, Bordetella pertussis, Staphylococcus spp., and Streptococcus spp. Gram-negative bacteria that typically also respond to such traditional therapies include Enterobacteria (e.g., Escherichia, Klebsiella, Proteus, Yersinia, Erwina), Salmonella, and Pseudomonas aeruginosa. Encapsulated bacteria resist phagocytosis and opsonization, often preventing more significant challenges to immune clearance, and include: Streptococcus spp., Haemophilus spp., Neisseria spp., Klebsiella spp., and Bacterioides fragillis.

Bacteria evade host defense by invading cells, thereby escaping serum antibodies and complement after specific stimulation. The removal of these infections almost completely depends on T lymphocyte-mediated immunity, and is particularly prone to becoming chronic infections. Specific examples include Salmonella (Salmonella typhi (S.typhi), S.choleraesuis, S.enteritidis), Legionella species (Legionella spp.), Listeria species (Listeria spp.), Brucella species (Brucella spp.) and Mycobacterium (Mycobacterium), including Mycobacterium tuberculosis (M.tuberculosis), Mycobacterium avium (M.avium) and Mycobacterium leprae (M.leprae).

Spirochete comprises that Treponema species (Treponema spp.), Borrelia species (Borrelia spp.) and Leptospira species (Leptospira spp.) are the bacterium that causes persistent and latent infection.Treponema palladium (Treponema palladium), the pathogen that causes disease syphilis, this disease is sexually transmitted disease, if not treated, may have serious pathological consequences.This disease is through different stage progress.Initial clinical stage is ulcer and the chancre of Treponema inoculation site.It is followed by the transfer distribution phase of spirochetemia and microorganism, and it continues to comprise infection and dissolving (being called the state of secondary syphilis) of repeated circulation.After the dissolving of secondary syphilis, disease enters asymptomatic latent period, and its result is tertiary syphilis, and this is serious often fatal state. Tertiary syphilis can manifest (i) in the heart as aortitis with aneurysm formation and subsequent aortic insufficiency, (ii) in the central nervous system (tabes dorsalis, paretic dementia), (iii) in the eye (interstitial keratitis), or (iv) in the ear (sensory deafness). Non-venereal forms resemble the clinical presentation of venereal forms but are primarily transmitted through direct contact and poor hygiene. They include yaws (T. pallidum subsp. pertenue), pinta (T. carateum), and bejel (T. pallidum subsp. endemicum).

Treatments for syphilis include penicillins (e.g., penicillin G), tetracycline, doxycycline, ceftriaxone, and azithromycin. The anti-PD-L1 antibodies of the present invention will most advantageously be administered for treatment during the latent stage of infection.

Lyme disease is caused by the bacterium Borrelia burgdorferi and is transmitted to humans through tick bites. The disease initially presents as a localized rash followed by flu-like symptoms including malaise, fever, headache, neck stiffness, and joint pain. Late manifestations may include migratory and polyarticular arthritis, neurological and cardiac involvement with cranial nerve palsies and radiculopathy, myocarditis, and arrhythmias. Some cases of Lyme disease become persistent, leading to irreversible damage similar to tertiary syphilis.

Current treatments for Lyme disease primarily involve the administration of antibiotics. Antibiotic-resistant strains can be treated with hydroxychloroquine or methotrexate. Antibiotic-resistant patients with neuropathic pain can be treated with gabapentin. Minocycline can be used for late-stage/chronic Lyme disease with neurological or other inflammatory manifestations. The anti-PD-L1 antibodies of the present invention are most advantageously administered for the treatment of latent infection.

Other forms of borreliosis, such as those caused by B. recurentis, B. hermsii, B. turicatae, B. parikeri., B. hispanica, B. duttonii, and B. persica, and leptospirosis (e.g., L. interrogans), typically resolve spontaneously unless blood titers reach concentrations that cause intrahepatic obstruction.

b. Viruses

For infections caused by viruses, the anti-PD-L1 antibodies of the present invention can be used simultaneously with, before, or after standard treatment for viral infections. Such standard treatments may vary depending on the type of virus, although in almost all cases, administration of human serum containing antibodies specific for the virus (e.g., IgA, IgG) is effective.

1) Influenza

Influenza infection causes fever, cough, myalgia, headache and malaise, which often occurs in seasonal epidemics. Influenza is also associated with a number of post-infectious conditions, such as encephalitis, myopericarditis, Goodpasture's syndrome and Reye's syndrome. Influenza infection also suppresses normal lung antibacterial defenses, so that patients who recover from influenza have an increased risk of developing bacterial pneumonia.

Influenza virus surface proteins show significant antigenic variation, which comes from mutation and recombination. Therefore, cytolytic T lymphocytes are the main tools for the host to clear the virus after infection. Influenza is divided into three main types: type A, type B and type C. Influenza A is unique in that it infects humans and many other animals (such as pigs, horses, poultry and seals) and is the main cause of widespread influenza. In addition, when a cell is infected with two different influenza A virus strains, the segmented RNA genomes of the two parental virus types mix during replication to produce hybrid replicons, resulting in new epidemic virus strains. Influenza B does not replicate in animals and therefore has less genetic variation, and influenza C has only a single serotype.

The most conventional therapy is a palliative to the symptoms caused by infection, and the host immune response actually removes the disease. However, some virus strains (for example, influenza A) can cause more serious diseases and death. Influenza A can be clinically and preventively treated, and this is carried out by administering the cyclic amine inhibitors amantadine (amantadine) and rimantadine (rimantadine) that suppress viral replication. However, the clinical application of these drugs is limited because relatively high side effect rate, their narrow antiviral spectrum (only influenza A) and the tendency that virus becomes resistant. Serum IgG antibodies for major influenza surface proteins, hemagglutinin and neuraminidase are administered to prevent lung infection, and mucosal IgA is needed to prevent upper respiratory tract and tracheal infection. The most effective treatment for influenza at present is to inoculate by administering the virus inactivated by formalin or β-propiolactone.

2) Measles virus

After an incubation period of 9–11 days, hosts infected with measles virus develop fever, cough, rhinitis, and conjunctivitis. Within 1–2 days, an erythematous, maculopapular rash develops, which rapidly spreads throughout the body. Because the infection also suppresses cell-mediated immunity, the host is at greater risk for bacterial superinfection, including otitis media, pneumonia, and postinfectious encephalomyelitis. Acute infection is associated with significant morbidity and mortality, particularly in malnourished adolescents.

Treatment for measles involves passive administration of pooled human IgG, which prevents infection in unimmunized subjects even when given a week after exposure. However, prior immunization with live, attenuated virus is the most effective treatment and prevention of the disease in over 95% of immunized individuals. Because the virus exists in only one serotype, a single immunization or infection generally results in lifelong protection against subsequent infection.

In a small percentage of infected hosts, measles can progress to SSPE, a chronic, progressive neurological disease caused by persistent infection of the central nervous system. SSPE is caused by measles virus clonal variants with defects that interfere with virion assembly and budding. For these patients, it is expected that the anti-PD-L1 antibodies of the present invention will revitalize T cells to assist in viral clearance.

3) Hepatitis B virus

Hepatitis B virus (HBV) is the most contagious blood-borne pathogen known. It is the leading cause of acute and chronic hepatitis and liver cancer, as well as lifelong chronic infection. Following infection, the virus replicates in liver cells, which then also shed the surface antigen HBsAg. Detection of excess levels of HBsAg in serum is used as the standard method for diagnosing hepatitis B virus infection. Acute infection can resolve or develop into a chronic, persistent infection.

Current treatments for chronic HBV include α-interferon, which increases the expression of type I human leukocyte antigen (HLA) on the surface of hepatocytes, thereby facilitating their recognition by cytotoxic T lymphocytes. In addition, the nucleoside analogs ganciclovir, famciclovir, and lamivudine have also shown some efficacy in treating HBV infection in clinical trials. Additional treatments for HBV include pegylated α-interferon, adenfovir, entecavir, and telbivudine. Although passive immunity can be conferred by parental administration of anti-HBsAg serum antibodies, vaccination with inactivated or recombinant HBsAg also confers resistance to infection. For therapeutic advantages, the anti-PD-L1 antibodies of the present invention can be combined with conventional treatments for hepatitis B virus infection.

4) Hepatitis C virus

Hepatitis C virus (HCV) infection can lead to a chronic form of hepatitis, resulting in cirrhosis. Although symptoms are similar to those of hepatitis B virus infection, unlike HBV, infected hosts can remain asymptomatic for 10-20 years. Treatment of HCV infection involves administration of a combination of interferon-alpha and ribavirin. A promising therapy for HCV infection is the protease inhibitor telaprevir (VX-960). Other treatments include anti-PD-1 antibodies (MDX-1106, Medarex), bavituximab (an antibody that binds to the anionic phospholipid phosphatidylserine in a B2-glycoprotein I-dependent manner, Peregrine Pharmaceuticals), anti-HPV viral envelope protein E2 antibodies (e.g., ATL 6865–Ab68+Ab65, XTL Pharmaceuticals), and (polyclonal anti-HCV human immunoglobulin). For therapeutic advantage, the anti-PD-L1 antibodies of the present invention can be combined with one or more of these therapies for hepatitis C infection.

Protease, polymerase, and NS5A inhibitors that can be used in combination with the anti-PD-L1 antibodies of the present invention to specifically treat hepatitis C infection include those identified in Table B below.

Table B

Hepatitis C protease and polymerase inhibitors

5) Human immunodeficiency virus (HIV)

HIV attacks CD4+ cells, including T-lymphocytes, monocyte-macrophages, follicular dendritic cells, and Langerhans cells, and depletes CD4+ helper/inducer cells. Consequently, the host acquires a severe deficiency in cell-mediated immunity. HIV infection leads to AIDS in at least 50% of individuals and is transmitted through sexual contact, administration of infected blood or blood products, artificial insemination with infected semen, exposure to needles or syringes containing blood, and from infected mothers to their babies during childbirth.

HIV infection can be asymptomatic or may develop an acute illness resembling mononucleosis—fever, headache, sore throat, malaise, and rash. Symptoms may progress to progressive immune dysfunction, including persistent fever, night sweats, weight loss, unexplained diarrhea, eczema, psoriasis, seborrheic dermatitis, herpes zoster, oral candidiasis, and oral hairy leukoplakia. Opportunistic infections with numerous parasitic helminths are common in patients whose infection progresses to AIDS.

HIV treatment includes antiviral therapy, including nucleoside analogs, zidovudine (AST), alone or in combination with didanosine or zalcitabine, dideoxyinosine, dideoxycytidine, lamidvudine, stavudine; reverse transcriptase inhibitors such as delavirdine, nevirapine, loviride, and protease inhibitors such as saquinavir, ritonavir, indinavir, and nelfinavir. For therapeutic advantages, the anti-PD-L1 antibodies of the present invention can be combined with conventional treatments for HIV infection.

6) Cytomegalovirus

Cytomegalovirus (CMV) infection is often associated with persistent, latent and recurrent infection. CMV infects and remains latent in monocytes and granulocyte-monocyte progenitor cells. The clinical symptoms of CMV include mononucleosis-like symptoms (i.e., fever, swollen glands, discomfort) and a tendency to develop an allergic rash to antibiotics. The virus is transmitted by direct contact. The virus is shed in urine, saliva, semen, and to a lesser extent in other body fluids. Transmission can also occur as follows: from an infected mother to her fetus or newborn and through blood transfusion and organ transplantation. CMV infection causes general damage to cellular immunity, which is characterized by an impaired embryonic response to nonspecific mitogens and specific CMV antigens, a reduced cytotoxic capacity and an elevated CD4+ lymphocyte CD8 lymphocyte count.

Treatment for CMV infection includes the antiviral ganciclovir, foscarnet, and cidovir, but these drugs are typically only given to immunocompromised patients. For therapeutic advantage, the anti-PD-L1 antibodies of the present invention can be combined with conventional treatments for CMV infection.

7) Epstein-Barr virus

Epstein-Barr virus (EBV) can establish persistent and latent infection and primarily attacks B cells. EBV infection leads to the clinical state of infectious mononucleosis, which includes fever, sore throat, often with exudates, systemic lymphadenopathy and splenomegaly. Hepatitis is also present, which can develop into jaundice.

Although typical treatment for EBV infection is palliative care, EBV has been linked to the development of certain cancers, such as Burkitt's lymphoma and nasopharyngeal cancer. Therefore, it is beneficial to eliminate viral infection before these complications develop. For therapeutic advantages, the anti-PD-L1 antibodies of the present invention can be combined with conventional treatments for EBV infection.

8) Herpes virus

Herpes simplex virus (HSV) is transmitted through direct contact with an infected host. Direct infection can be asymptomatic but generally results in blisters containing infectious particles. The disease manifests as cycles of active disease, in which lesions appear and disappear as the virus latently infects ganglia and subsequently erupts. Lesions can be on the face, genitals, eyes, and/or hands. In some cases, infection can also lead to encephalitis.

Treatment of herpes infections primarily involves resolving symptomatic outbreaks, including systemic antiviral drugs such as acyclovir (e.g., valaciclovir), famciclovir, and penciclovir, and topical agents such as tromantadine and zilactin. Clearing latent herpes infections is clinically beneficial. For therapeutic advantages, the anti-PD-L1 antibodies of the present invention can be combined with conventional treatments for herpes virus infections.

9)HTLV

Human T-lymphotrophic virus (HTLV-1, HTLV-2) is transmitted through sexual contact, breastfeeding or exposure to contaminated blood. The virus activates a subtype of _T cells called Th1 cells, causing their transitional proliferation and overproduction of Th1-related cytokines (e.g., IFN-γ and TNF-α). This in turn leads to the suppression of Th2 lymphocytes and the production of reduced Th2 cytokines (e.g., IL-4, IL-5, IL-10 and IL-13), resulting in a reduced ability of the infected host to establish sufficient immune responses against invading organisms, which require Th2-dependent responses to clear (e.g., parasitic infections, the production of mucosal and humoral antibodies).

Opportunistic infections caused by HTLV infection lead to bronchiectasis, dermatitis, and superinfection with Staphylococcus spp. and Strongyloides spp., leading to death due to polymicrobial sepsis. HTLV infection can also directly lead to adult T-cell leukemia/lymphoma and progressive demyelinating upper motor neuron disease, known as HAM/TSP. Clearance of latent HTLV infection is clinically beneficial. For therapeutic advantages, the anti-PD-L1 antibodies of the present invention can be combined with conventional treatments for HTLV infection.

10) HPV

Human papillomavirus (HPV) primarily affects keratinocytes and occurs in two forms: cutaneous and genital. Transmission is believed to occur through direct contact and/or sexual activity. Both cutaneous and genital HPV infection can lead to warts and latent and sometimes recurrent infections that are controlled by the host's immune system, which controls symptoms and blocks the development of warts, but allows the host to spread the infection to others.

HPV infection can also lead to certain cancers, such as cervical cancer, anal cancer, vulvar cancer, penile cancer, and oropharyngeal cancer. There is no known cure for HPV infection, but current treatment is topical imiquimod, which stimulates the immune system to attack the affected area. Clearing latent HPV infection is clinically beneficial. For therapeutic advantages, the anti-PD-L1 antibodies of the present invention can be combined with conventional treatments for HPV infection.

c. Fungi

Fungal infections (or mycoses) can result in primary infections or can result in opportunistic colonization of hosts with compromised immune systems by endogenous flora. Immunity to mycoses appears to be primarily cellular, involving neutrophils, macrophages, lymphocytes, and possibly natural killer (NK) cells. Mycoses are generally not directly killed by antibodies and complement. Systemic invasive fungal diseases resulting from primary infections include blastomycosis, coccidioidomycosis, histoplasmosis, and paracoccidioidomycosis. For chronic infections resulting from fungal infections, the anti-PD-L1 antibodies of the present invention can be administered before, simultaneously with, or subsequently to any conventionally known treatment for these mycoses.

Blastomycosis, caused by Blastomyces dermatitis, is acquired by inhalation and produces a primary lung infection or hematogenously disseminated disease, primarily affecting the skin, bones, and male urogenital tract. Initial exposure may be asymptomatic, or it may produce influenza-like symptoms. The disease may manifest as a chronic, painless form. The disease is also associated with impaired immunity, such as in patients with AIDS. Conventional treatments for Blastomyces dermatitis infections include itraconazole, ketoconazole, or intravenous amphotericin B.

Coccidioidomycosis, caused by the bacterium Coccidioides immitis, is acquired by inhalation and results in primary lung infection, progressive lung disease, or hematogenous dissemination, primarily involving the skin, subcutaneous tissue, bones, joints, and meninges. Initial exposure may be asymptomatic (60%) or associated with influenza-like symptoms. Pneumonia, pleuritis, and pulmonary cavitation may occur. Metastatic manifestations include skin lesions, including nodules, ulcers, sinus tracts, and verrucous granulomas arising from deeper sites, bones, joints, tendons, and meninges, including meningitis. Disease is also associated with impaired immunity, such as in patients with AIDS. Treatment of coccidioidomycosis includes ketoconazole, itraconazole, and fluconazole, particularly for long-term maintenance treatment of nonmeningeal disease. Meningeal forms are usually treated with intrathecal amphotericin B.

Histoplasmosis, caused by Histoplasma capsulatum, is an inhalation-acquired disease of the reticuloendothelial system in which small yeasts reside in macrophages. It can produce primary lung infection, progressive lung disease, or hematogenous dissemination, primarily involving the reticuloendothelial system, mucosal surfaces, and adrenal glands. Reactivation of latent infection often occurs in immunocompromised patients, such as those with AIDS. Initial exposure may be asymptomatic or associated with cold-like symptoms, including pneumonia, pleurisy, pulmonary cavitation, and mediastinal adenopathy. Metastatic sites include the reticuloendothelial system (hepatosplenomegaly, lymphadenopathy, anemia, leukopenia, and thrombocytopenia), mucosal membranes (oronasal and pharyngeal ulcers), gastrointestinal tract (malabsorption), and adrenal insufficiency. Although most primary infections resolve spontaneously, when associated with impaired immunity (as in patients with AIDS), relapses occur and are often associated with hematogenous pneumonia, ARDS, disseminated intravascular coagulation (DIC), hematogenously distributed papulopustules, and meningitis. Histoplasmosis is treated with amphotericin B (particularly in acute cases with hematogenous spread in immunocompromised patients), itraconazole, and ketoconazole.

Paracoccidioidomycosis, caused by Paracoccidioides brasiliensis, is an inhalation-acquired fungal infection that can cause primary pulmonary infection or hematogenous dissemination, primarily involving the skin, mucous membranes, reticuloendothelial system, and adrenal glands. The infection may initially be asymptomatic and quiescent, then reactivate. Treatment of this infection is with ketoconazole, itraconazole, and sulfonamides.

Systemic invasive fungal diseases are caused by opportunistic pathogens that occur in immunocompromised hosts and include candidiasis, cryptococcosis, aspergillosis, mucomycosis, and pneumocystosis. By enhancing the immune response in an impaired immune system, the anti-PD-L1 antibodies of the present invention may also have therapeutic value in treating these conditions, especially when combined with conventional therapies.

Candidiasis (caused by Candida albicans, C. tropicalis, C. glabrata), cryptococcosis (caused by Cryptococcus neoformans), aspergillosis (caused by Aspergillus flavus, A. fumigatus, A. tereus, and A. niger), and mucormycosis (caused by Rhizopus arrhizus, Rhizomuco, Absidia, Cunninghamella, Mortierella, Saksenaea spp.) can be treated with one or more of the following: imidazoles, ketoconazole, itraconazole, fluconazole, amphotericin B with or without flucytosine. Pneumocystis (caused by penumocystis carnii), recently reclassified from a protozoan to a fungus, is treated with trimethoprim-sulfamethoxole (TMP-SMZ) and intravenous pentamidine isethionate, as well as dapsone, TMP-dapsone, trimetrexate, clindamycin-primaquine, and atovagnone.

Microsporidiosis is caused by parasites of the order Microsporidia, recently reclassified from protozoa to fungi. They are single-celled organisms with spindles instead of mitochondria. Organisms that can cause disease in humans include Enterocytozoon bieneusi, Encephalitozoon hellem, Encephalitozoon intestinalis, Encephalitozoon cuniculi, Pleistophora spp., Trachipleistophora hominis, Trachipleistophora anthropophthera, Nosema connori, Nosema ocularum, Brachiola vesicularum, Vittaforma corneae, Microsporidium ceylonensis, Microsporidium africanum, and Brachiola algerae.

It is believed that infection is transmitted to humans by direct contact with animals, contaminated water, or another infected host. After infecting host cells, the sporoplasm grows, divides, or forms a multinucleate plasmodium, which can have a complex life cycle, including both asexual and sexual reproduction. Microsporidian infection is often characterized by continuous generations of autoinfection and a chronic, debilitating disease.

Clinical manifestations of disease vary depending on the species and host immune status and include conjunctivitis (e.g., V. corneae), chronic diarrhea, malabsorption, and emaciation (e.g., E. bieneusi, E. intestinalis).

Treatment for ocular, intestinal, and disseminated microsporidiosis includes administration of albendazole. Topical fumagillin is also effective for treating microsporidial keratoconjunctivitis. Other medications include antihelminthics (e.g., albendazole), antibiotics (e.g., fumagillin), immunomodulators (e.g., thalidomide), and antiprotozoal agents (e.g., metronidazole).

d. Protozoa

Diseases caused by parasites, such as malaria, schistosomiasis, and leishmaniasis, are among the most prevalent and important health problems in developing countries. These diseases are particularly challenging because they evade host immunity in multiple ways, including: 1) living inside host cells (e.g., Leishmania), 2) rapidly changing surface antigens (e.g., trypanosomes), and 3) "disguising" themselves as host cells by displaying host antigens (e.g., schistosomiasis). The use of immunosuppressive drugs in the treatment of cancer and in conjunction with organ transplantation, as well as in the global AIDS epidemic, can reactivate latent or subclinical infections from Plasmodium spp., Toxoplasma spp., Leishmania spp., Cryptosporidium spp., Trypanosoma spp., and helminths.

For chronic infections due to protozoan parasites, the anti-PD-L1 antibodies of the invention can be administered in conjunction with, before, or after standard antiprotozoal therapy.

Malaria is caused by parasites of the genus Plasmodium (e.g., P. ovale, P. malariae, P. falciparum, P. vivax), and the infectious cycle begins with sporozoites, which develop in the intestines of female anopheline mosquitoes. Upon transmission to humans, these sporozoites invade liver cells and replicate without inducing an inflammatory response. The progeny of these organisms, called merozoites, then invade red blood cells and initiate the clinical stage of the disease, typically characterized by fever and chills. In areas of the world where the infection is endemic, almost all residents carry a continuous low-level chronic infection of low to moderate pathogenicity, with increased levels of IgG antibodies providing protection from merozoites entering red blood cells.

Currently available antimalarial drugs for the treatment and prevention of clinical disease include: Artemether-lumefantrine (treatment, e.g., and ), artesunate-amodiaquine (treatment), artesunate-mefloquine (treatment), artesunate-Sulfadoxine/pyrimethamine (treatment), atovaquone-proguanil, (treatment and prevention, e.g., ), quinine ( quinine (treatment), chloroquine (treatment and prevention), cotrifazid (treatment and prevention), doxycycline (treatment and prevention), mefloquine (treatment and prevention, for example, ), primaquine (treatment of P. vivax and P. ovale only, not for prevention), proguanil (prevention), sulfadoxine-pyrimethamine (treatment and prevention), hydroxychloroquine (treatment and prevention, for example, ).

The anti-PD-L1 antibodies of the present invention may be particularly useful in therapies to assist in the clearance of malaria parasites by revitalizing anergic T cells.

Toxoplasmosis, caused by parasites of the genus Toxoplasma, is often asymptomatic, but a small percentage develop clinical disease, which can range from benign acute lymphadenopathy to fatal infection of the central nervous system. Sources of infection include cysts in raw or partially cooked pork or lamb and oocysts transferred in the feces of infected cats. Infection in humans usually occurs through the gastrointestinal tract, where the protozoa can penetrate and multiply (as tachyzoites) in almost every cell of the body. These tachyzoites can produce cysts filled with tiny, slow-growing infectious bodies (bradyzoites) that can survive for a long time, leading to latent chronic infection. Hosts with compromised immune systems, such as those taking immunosuppressive drugs or suffering from HIV, are particularly susceptible to toxoplasmosis.

Drugs used to treat primary toxoplasmosis include the following: pyrimethamine, with or without antibiotics (for example, sulfadiazine, clindamycin, spiramycin, and minocycline). Latent toxoplasmosis can be treated with the antibiotic atovaquone with or without clindamycin.

Leishmaniasis, caused by parasites of the genus Leishmania, infects macrophages in the skin and internal organs and is transmitted to humans by sand flies. Because there is little or no specific serum antibody, cell-mediated immunity through activated T cells appears to be the key pathway for clearing the infection. Old World leishmaniasis, also known as tropical sore, is caused by several species of Leishmania: L. tropicala, L. major, and L. aethiopica. New World leishmaniasis is caused by various subspecies of L. Mexicana and L. braziliensis. These parasites induce a strong cell-mediated immune response, but the clinical outcome is also due in part to the host's response. If the host mounts a suppressed or inadequate cell-mediated response, the result is a disseminated chronic cutaneous leishmaniasis with little hope of spontaneous recovery (e.g., Leishmania ethiopiana, Leishmania mexicana). If the host mounts an excessive cell-mediated response, the response is lupus-like or recidiva leishmaniasis, with persistent, non-ulcerated lymphoid nodules appearing at the margins of the primary lesion (e.g., Leishmania tropicalis). Recidiva leishmaniasis can appear 1 to 10 years after the initial injury. There are two forms of the disease, cutaneous and visceral. The cutaneous form presents with skin lesions, and cell-mediated immunity is critical for clearance. In the visceral form, cell-mediated immunity is inadequate or absent, and the disease is clinically manifested by polyclonal B-cell hypergammaglobulinemia, leukopenia, splenomegaly, and increased TNF-α production.

Miltefosine (eg, LEISHINE(R)) and paramyocin are currently available therapies for cutaneous and visceral leishmaniasis.

Cryptosporidiosis (Crytosporidiosis), caused by infection with protozoa of the genus Cryptosporidia, is caused by direct contact with fecal excretions of infected hosts. Infection of the intestinal mucosal tissue can lead to diarrhea. The disease typically manifests as an acute infection, but it can become chronic, particularly in immunocompromised individuals. Treatment is generally palliative, particularly hydration, but paromomycin, azithromycin, and serum Ig (e.g., ) have been successfully used to clear the infection.

Trypanosomiasis, caused by Trypanosoma parasites (e.g., T. Brucei subsp., T. rhodesiense subsp.), infects humans and cattle through the bite of the tsetse fly. The challenge of this pathogen arises from successive generations of a population displaying different surface antigens. Infection is characterized by elevated levels of nonspecific and nonprotective serum immunoglobulins.

Treatment of trypanosomiasis includes intravenous administration of pentamidine (for T. b. gambiense), intravenous suramin (for T. b. rhodesiense), eflornithine, melarsoprol with or without nifurtimox.

Helminth infections caused by flukes (eg, Schistosoma spp.), cestodes, and nematodes all have a general immune response of eosinophilia and reactive antibodies that is T cell dependent.

Schistosomiasis (also known as bilharzia), caused by Schistosoma mansoni, S. japonicum, S. haematobium, and S. mekongi, begins its life cycle in water as eggs that hatch into ciliated larvae that penetrate snails and produce multiple generations of sporocysts. These, in turn, produce forked-tailed cercariae that can infect the bloodstream of a human host as shistosomula, initially migrating to the lungs and then to the liver. These flukes eventually pair up, mate, and lay eggs in the mesenteric venules. Although many of these eggs travel to the intestines and are expelled, some become entrapped in the submucosa, the portal vein of the liver, and other organs of the body. Granulomatous inflammation associated with the entrapped eggs is a defining symptom of chronic schistosomiasis.

Treatment of schistosomiasis includes administration of praziquantel antimony, hydroxyaminoquine (Schistosoma mansoni), and

Tapeworm infections can be divided into two groups. One group is intestinal-dwelling adult tapeworms, such as Diphyllobothrium latum and Taenia saginata, which have a limited, non-humoral immune response. The second group describes migratory tissue-encapsulated larval tapeworms, such as Hymenolepis nana, Echinococcus granulosus, and Taenia solium, which induce a strong parenteral host response and protective serum antibodies. The most serious tapeworm infection in humans is echinococcosis, which can lead to the formation of hydatid cysts when implanted in the liver, lungs, brain, kidneys, or other parts of the body.

Treatment of hydatid disease includes administration of metronidazole, albendazole (thiabendazole) and surgical interventions such as removal, aspiration, marsupialization, or omentopexy.

Nematodes are the most common, diverse, and widespread helminths that infect humans, causing conditions such as trichnosis, ascariasis, filariosis, and strongylodiosis. Trichinosis, caused by the nematode Trichinella spiralis, can result from ingesting larvae of Trichinella spiralis in raw or partially cooked meat, such as pork. In humans, infection induces a strong humoral response, with elevated IgM followed by IgG production, followed by rapid elimination of the helminth by antibody damage by T lymphocytes.

The only known treatment that kills adult worms in the intestine is thiabendazole, and there are no known treatments that kill the larvae.

Ascaris, also known as giant roundworms (Ascaris lumbricoides), are common parasites in humans that are caused by ingesting fecal contamination. Although patients can remain asymptomatic for very long periods as the larval stages travel through the body, they can cause damage to internal organs, peritonitis and inflammation, enlargement of the liver or spleen, toxicity, and pneumonia.

Treatment for roundworms includes administration of mebendazole (e.g., ), piperazine, pyrantel pamoate (e.g., Pin-Pin-), albendazole, thiabendazole with or without piperazine, hexylresorcinol, santonin, and wormwood oil. The anti-PD-L1 antibodies of the present invention can be administered in combination with, before, or after these therapies for treating roundworms.

Filariasis (filariosis), caused by filarial nematodes (filarid nematodes), is introduced into humans through insect vectors. Onchocerca volvulus (Onchocerca volvulus), causes onchoceriasis (onchoceriasis) or river blindness (riverblindness), and is transmitted through the bites of black flies. The infective larvae themselves live subcutaneously, develop into adults, induce a fibrogenic host response, and shed a large number of cercariae, which disperse subcutaneously and pass through the eyes, further inducing keratitis or retinitis, which then causes the cornea to become opaque. Lymphatic filariasis is caused by infection with Brugia species (Brugia spp.) and Wuchereria species (Wuchereria spp). Over a period of time, scarring of lymphatic tissue (particularly in the groin) can hinder drainage, leading to the disfiguring condition elephantiasis.

The main treatment for filariasis is administration of the antibiotics ivermectin, albendazole, and diethylcarbamazine citrate (DEC,) with or without ivermectin and albendazole. Other treatment options include doxycycline, which kills the commensal bacteria Wolbachia.

Strongyloidiasis, caused by parasites of the genus Strongyloides (e.g., S. stercoralis, S. fülleborni), is a disease transmitted to humans via fecal contamination of soil. They can have both a free life cycle (rhabditiform larvae mature into adults) and a parasitic life cycle (filariform larvae mature into adults), the latter of which penetrates the skin, travels to the lungs, then the pharynx, and finally resides in the intestines. Autoinfection with Strongyloides is also known to occur, which is essentially a superinfection with successive generations of filariform larvae.

Infection may be asymptomatic or may be characterized by gastrointestinal pain and diarrhea, pulmonary Loeffler syndrome (i.e., eosinophilia), and urticaria. Blood eosinophilia may also be present. Because persistent infection with Strongyloides nematodes can mimic peptic ulcers, gallbladder disease, and Crohn's disease, misdiagnosis is common. It is particularly problematic in immunocompromised hosts.

Known treatments for strongyloidiasis are ivermectin, albendazole or thiabendazole, but because these drugs only kill adult worms, repeated administration is necessary.

e. Vaccination

Vaccination or administration of antigenic substances are routinely used to prevent or improve the effects of pathogen infection to induce immunity to disease. Enhanced host immunity can be used on undesirable antigens that are not only found on infectious pathogens, but also on host tissues infected with diseases (e.g., cancer). Traditionally, vaccines are derived from weakened or dead whole pathogens, but they can also be peptides representing epitopes on intact pathogens that are specifically recognized by human class I or class II major histocompatibility complex (MHC) molecules. Peptide antigens of particular interest are those that are specifically recognized by T cells.

Recently, it has been shown that combining therapeutic vaccination with PD-L1 blockade on exhausted CD8+ T cells results in enhanced function and viral control in a mouse model of chronic infection. Ha et al., J. Exp. Med. 205(3):543-555 (2008). Thus, the anti-PD-L1 antibodies described herein can also be combined with antigen immunization (e.g., administered before, simultaneously with, or after) to treat infections (e.g., acute and chronic) caused by viral, bacterial, fungal, or protozoal invasion, as well as tumor immunity.

G. Drug dosage:

The dosage and desired drug concentration of the pharmaceutical compositions of the present invention may vary depending on the specific intended use. Determining an appropriate dosage or route of administration is well within the capabilities of those of ordinary skill. Animal studies provide reliable guidance for determining effective doses for human therapy. Interspecies conversion of effective doses can be performed according to the principles set forth in Mordenti, J. and Chappell, W. "The Use of Interspecies Scaling in Toxicokinetics," in Toxicokinetics and New Drug Development, Yacobi et al., eds., Pergamon Press, New York 1989, pp. 42-46.

When administering the polypeptides or antibodies described herein in vivo, normal dosage amounts may range from about 10 ng/kg to about 100 mg/kg of mammalian body weight or more per day, preferably about 1 mg/kg/day to 10 mg/kg/day, depending on the route of administration. Guidance on specific dosages and methods of administration is provided in the literature; see, for example, U.S. Patent Nos. 4,657,760; 5,206,344; or 5,225,212. It is within the scope of the present invention that different formulations are effective for different treatments and different diseases, and that administration intended to treat a particular organ or tissue may necessitate administration in a different manner than that intended to treat another organ or tissue. Furthermore, dosing may be performed by one or more separate administrations, or by continuous infusion. For repeated administrations over several days or longer, depending on the circumstances, treatment is continued until the desired suppression of disease symptoms occurs. However, other administration schedules are also useful. The progress of treatment can be readily monitored by conventional techniques and assays.

H. Administration of the Formulation

The formulations of the present invention, including but not limited to reconstituted formulations and liquid formulations, are administered to a mammal, preferably a human, in need of treatment with an anti-PD-L1 antibody according to known methods, for example, by intravenous administration as a bolus, or by continuous infusion over a period of time by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical or inhalation routes.

In a preferred embodiment, the formulation is administered to a mammal by subcutaneous administration (e.g., under the skin). To this end, the formulation can be injected using a syringe. However, other devices for administering the formulation are available, such as injection devices (e.g., INJECT-EASE ^™ and GENJECT ^™ devices); injection pens (e.g., GENPEN ^™ ); automatic injection devices, needle-free devices (e.g., MEDIJECTOR ^™ and BIOJECTOR ^™ ) and subcutaneous patch delivery systems.

In a specific embodiment, the present invention relates to a kit for a single-dose administration unit. Such a kit includes a container for an aqueous formulation of a therapeutic protein or antibody, including a single-chamber or multi-chamber prefilled syringe. Exemplary prefilled syringes are available from Vetter GmbH, Ravensburg, Germany.

The appropriate dosage of the protein ("therapeutically effective amount") will depend on, for example, the condition to be treated, the severity and course of the condition, whether the protein is being administered for preventive or therapeutic purposes, previous treatments, the patient's medical history and response to the anti-PD-L1 antibody, the form of the formulation used, and the judgment of the attending physician. The anti-PD-L1 antibody is suitably administered to the patient at one time or over a series of treatments, and can also be administered to the patient at any time since diagnosis. The anti-PD-L1 antibody can be administered as the sole treatment or in combination with other drugs or treatments useful in treating the current condition.

For anti-PD-L1 antibodies, an initial candidate dose range may be about 0.1-20 mg/kg administered to the patient, which may be administered in one or more separate administrations. However, other administration schedules are also useful. The progress of such treatment can be easily monitored by conventional techniques.

I. Products

In another embodiment of the present invention, a product comprising the formulation is provided, preferably with instructions for use thereof. The product comprises a container. Suitable containers include, for example, bottles, vials (e.g., double-chamber vials), syringes (e.g., single-chamber or double-chamber syringes) and test tubes. The container can be made of various materials such as glass or plastic. The container is filled with the formulation. A label located on or associated with the container may indicate instructions for reconstitution and/or use. The label may further indicate that the formulation is useful for subcutaneous administration or is for subcutaneous administration, and/or is used to treat T cell dysfunction disorders. The container with the formulation may be a reusable vial that allows for repeated administration (e.g., 2-6 administrations) of the reconstituted formulation. The product may further include a second container comprising a suitable diluent (e.g., BWFI). When mixing the diluent and the lyophilized formulation, the final protein concentration in the reconstituted formulation is generally at least 50 mg/ml. The product may further include other required materials from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

The present invention will be more fully understood by reference to the following examples. However, these examples should not be considered as limiting the scope of the present invention. All references disclosed herein are expressly incorporated herein by reference.

In another embodiment, the present invention provides an article of manufacture comprising a formulation as described herein for administration in an autoinjector device. An autoinjector can be described as an injection device that delivers its contents without requiring additional action by the patient or the user upon activation. They are particularly suitable for self-administration of therapeutic formulations when a constant administration rate is required and the administration time is relatively long.

Example 1

Identification of anti-PD-L1 antibodies from phage libraries

Library sorting and screening to identify anti-PD-L1 antibodies

Human (R&D Systems, cat#156-B7) and mouse (R&D Systems, cat#1019-B7) PD-L1-Fc fusions were used as antigens for alternating library sorting. Specifically, the phage library was first sorted against human antigens and then sorted against mouse, human, and mouse antigens in three subsequent rounds. Nunc 96-well immunoplates were coated with target antigens (10 μg/ml) overnight at 4°C and blocked with phage blocking buffer PBST (phosphate-buffered saline (PBS) with 1% (w/v) bovine serum albumin (BSA) and 0.05% (v/v) tween-20) at room temperature for 1 hour. Antibody phage libraries VH (see, e.g., Lee et al., J. Immunol. Meth. 284 : 119-132, 2004) and VH/VL (see Liang et al., J. Mol. Biol. 366 : 815-829, 2007) were added individually to antigen plates and incubated overnight at room temperature. The next day, the antigen-coated plates were washed ten times with PBT (PBS containing 0.05% Tween-20), and the bound phage were eluted with 50mM HCl and 500mM NaCl for 30 minutes and neutralized with an equal volume of 1M Tris base (pH 7.5). The recovered phage were amplified in E. coli XL-1 Blue cells. In subsequent selection rounds, the incubation time of the antibody phage with the antigen-coated plates was reduced to 2-3 hours, and the stringency of the plate washing was gradually increased.

After 4 rounds of panning, significant enrichment was observed. 96 clones were selected from each of the VH and VH/VL library sorting to determine whether they specifically bind to both human and mouse PD-L1-Fc. The variable regions of these clones were sequenced by PCR to identify unique sequence clones.

The parental clones of interest were reformatted into IgG by cloning the _VL and _VH regions of individual clones into LPG3 and LPG4 vectors, respectively (Lee et al., supra), transiently expressing them in mammalian CHO cells, and purifying them by protein A column. Thirteen phage antibodies were evaluated for their ability to block the interaction between soluble PD-1-Fc fusion protein and human or murine PD-L1 expressed in 293 cells (IC 50 values are indicated in the upper half of Table 1). YW243.55 was selected for subsequent affinity maturation to improve its affinity for both human and murine PD-L1, having the lowest IC ₅₀ for blocking human PD-L1 binding to PD-1 (Table 1). Antibodies with comparable cross-reactivity against both primate and murine species (while also retaining affinity for humans) would provide therapeutics with enhanced value, as the same antibodies well characterized in experimental models could be used in human clinical trials. This avoids the uncertain results that come from using model-specific surrogates.

Construction of affinity-improved libraries for clones derived from _VH libraries

Phagemid pW0703 (derived from phagemid pV0350-2b (Lee et al., J. Mol. Biol. 340 , 1073-1093 (2004)), containing stop codons (TAA) in all CDR-L3 positions and displaying monovalent Fab on the surface of M13 phage) was used as a library template for grafting heavy chain variable domains ( _VH ) of clones of interest from the _VH library for affinity maturation. Hard and soft randomization strategies were used for affinity maturation. For hard randomization, a light chain library with selected positions of three light chain CDRs was randomized using amino acids designed to mimic natural human antibodies, and the designed DNA degeneracy was described in Lee et al. (J. Mol. Biol. 340, 1073-1093 (2004)). For soft randomization, residues at positions 91-94 and 96 of CDR-L3, 28-31 and 34-35 of CDR-H1, 50, 52 and 53-58 of CDR-H2, and 95-99 and 100A of CDR-H3 were targeted; and two different combinations of CDR loops (L3/H1/H2 and L3/H3) were selected for randomization. To achieve the soft randomization scenario, which introduces approximately 50% mutation rate at the selected sites, mutagenic DNA was synthesized using a 70-10-10-10 base mixture that supports wild-type nucleotides (Gallop et al., Journal of Medicinal Chemistry 37:1233-1251 (1994)).

Phage sorting with improved affinity

The previously identified phage clones were subjected to a first round of plate sorting, followed by five or six rounds of solution sorting. Each of the libraries was sorted separately for human and mouse PD-L1-Fc (R&D Systems, cat.#156-B7, cat#1019-B7, respectively). For the human PD-L1-Fc target, in the first round of plate sorting, the three libraries were sorted for 2 hours each on a target-coated plate (NUNCplate) at room temperature using approximately 3 OD/ml of phage input in 1% BSA and 0.05% Tween 20. After the first round of plate sorting, solution sorting was performed to increase the stringency of selection. For solution sorting, 10.D./ml of phage propagated from the first round of plate sorting was incubated with 20 nM biotinylated target protein (concentration based on the _IC50 value of the parental clone phage) in 100 μL of buffer containing 1% Superblock (Pierce Biotechnology) and 0.05% Tween-20 at room temperature for 30 minutes. The mixture was further diluted 10X with 1% Superblock and 100 μL/well was applied to neutravidin-coated wells (5 μg/ml) with gentle shaking at room temperature for 15 minutes to allow biotinylated target to bind to phage. The wells were washed 10X with PBS-0.05% Tween-20. To determine background binding, control wells containing phage with non-biotinylated target were captured on neutravidin-coated plates. The bound phage were eluted with 0.1N HCl for 20 minutes, neutralized with 1/10 volume of 1M Tris pH-11, titrated and propagated for the next round. Thereafter, five more rounds of solution sorting were performed together by increasing the selection stringency by two methods. The first round was an on-rate selection, which was performed by reducing the concentration of the biotinylated target protein from 4nM to 0.5nM, and the second round was an off-rate selection, which was performed by adding an excess of non-biotinylated target protein (100-2000 times more) to compete out the weaker binders, both at room temperature or 37°C. In addition, the phage input was reduced (0.1-0.5OD/ml) to reduce background phage binding. For the mouse PD-L1-Fc target, the phage sorting method was similar to the method described above for the human PD-L1Fc antigen, with slight modifications. Specifically, 100 nM biotinylated murine PD-L1-Fc was used for solution panning immediately after the first round of plate panning. In the following four rounds of solution panning, the biotinylated target was reduced from 10 nM to 1 nM, and a 200-500-fold excess of non-biotinylated target was added at room temperature.

Affinity matured clones were then further screened using the high throughput affinity screening ELISA procedure described in the Examples below.

High-throughput affinity screening ELISA (single-spot competition)

Colonies were picked from the seventh and sixth rounds of screening for human and mouse PD-L1 targets, respectively. The colonies were grown overnight at 37°C in 150 μL/well of 2YT medium containing 50 μg/ml carbenicillin and 1E10/ml KO7 in a 96-well plate (Falcon). From the same plate, colonies of XL-1-infected parental phage were picked as controls. 96-well Nunc plates were coated with 100 μL/well of human and mouse PD-L1-Fc proteins (2 μg/ml) each in PBS at 4°C overnight or at room temperature for 2 hours. The plates were blocked with 65 μL of 1% BSA for 30 minutes and 40 μL of 1% Tween 20 for another 30 minutes.

Phage supernatants were diluted 1:10 in ELISA (enzyme-linked immunosorbent assay) buffer (PBS containing 0.5% BSA, 0.05% Tween-20) with or without 10 nM target protein in a total volume of 100 μL and incubated in F plates (NUNC) at room temperature for at least 1 hour. 75 μL of the mixture with or without target protein was transferred side by side to the target protein-coated plate. The plate was gently shaken for 15 minutes to allow unbound phage to be captured on the target protein-coated plate. The plate was washed at least five times with PBS-0.05% Tween-20. Binding was quantified by adding horseradish peroxidase (HRP)-conjugated anti-M13 antibody to ELISA buffer (1:5000) and incubating at room temperature for 30 minutes. The plate was washed at least five times with PBS-0.05% Tween-20. Afterwards, 100 μL/ _well of a 1:1 ratio of 3,3',5,5'-tetramethylbenzidine (TMB) peroxidase substrate and peroxidase solution B ( _H2O2 ) (Kirkegaard-Perry Laboratories (Gaithersburg, MD)) was added to the wells and incubated at room temperature for 5 minutes. The reaction was terminated by adding 100 μL of 1M phosphoric acid ( _H3PO4 ) to each well and allowed to incubate at room temperature for 5 minutes. The OD (optical density) of the yellow color in each well was determined at 450 nm using a standard ELISA plate reader. _The reduction in OD (%) was calculated by the following equation.

OD _450nm reduction (%) = [(OD _450nm of wells containing competitor)/(OD _450nm of wells without competitor)] x 100

Clones with a OD _450nm reduction (%) of less than 50% for both human and murine targets compared to the OD _450nm reduction (%) of the parental phage wells (100%) were selected for sequence analysis. Unique clones were selected from the phage preparations to determine the binding affinity (phage IC ₅₀ ) for both human and murine PD-L-Fc by comparison with the parental clone.

Material

hPD-1-Fc, hPD-L1-Fc, hB7.1-Fc, mPD-1-Fc, mPD-L1-Fc, and mB7.1 were purchased from R&D Systems. 293 cells expressing hPD-L1 were generated at Genentech using conventional techniques. F(ab′) ₂ goat anti-human IgG Fc was purchased from Jackson ImmunoResearch Laboratories.

Protein conjugation

PD-1-Fc and B7.1-Fc proteins were biotinylated using EZ-Link sulfo-NHS-LC-LC-biotin (Pierce) at room temperature for 30 minutes as described by the manufacturer. Excess unreacted biotin was removed using Quick Spin High Capacity Columns, G50-Sephadex (Roche) as described by the manufacturer.

F(ab') ₂ goat anti-human IgG Fc was ruthenium-labeled using MSD Sulfo-label NHS-Ester (Meso Scale Discovery) as described by the manufacturer and excess non-reactive Sulfo-label was removed using a Quick Spin High Capacity Column, G50-Sephadex.

ECL cell binding assay for testing phage antibodies

The antibody concentration that results in 50% inhibition of hPD-1-Fc binding to 293 cells expressing hPD-L1 (IC ₅₀ ) was measured by electrochemiluminescent (ECL) cell binding assay. 293 cells expressing hPD-L1 were washed with phosphate-buffered saline (PBS) and seeded at 25,000 cells per well in 25 μL PBS on a 96-well high-binding plate (Meso Scale Discovery). The plate was incubated at room temperature to allow cells to attach to the carbon surface of the plate. 25 μL of 30% FBS was added to each well and the plate was incubated for 30 minutes with gentle shaking to block nonspecific binding sites. The plate was washed three times with PBS on an ELISA microplate washer (ELx405 Select, Bio-Tek Instruments) under gentle dispersion and aspiration. Excess PBS in the wells was removed by blotting the plate on a paper towel. 12.5 μL of 2X antibody concentration in 3% FBS in PBS (assay buffer) was added to each well. Then, 12.5 μL of 4 μg/mL (2X concentration) hPD-1-biotin in assay buffer was added and the plate was incubated for one hour with gentle shaking. The plate was washed three times with PBS in a microplate washer and blotted dry on a paper towel. 25 μL of 2 μg/mL streptavidin-ruthenium (Meso Scale Discovery) was added and incubated in assay buffer at room temperature with gentle shaking for 30 minutes. The plate was washed three times with PBS in a microplate washer and blotted dry on a paper towel. 150 μL of 1X MSD reading buffer (Meso Scale Discovery) without surfactant was added. Luminescence light was read at 620 nm on a Sector Imager 6000 reader (Meso Scale Discovery). ECL values were analyzed using a four-parameter nonlinear least squares fit with the test antibody concentrations used in the assay to obtain _IC50 values for each competitor in the assay.

Results and discussion:

Fifteen unique phage antibodies derived from YW243.55 that bound both human and murine PD-L1 were selected and reformatted into full-length IgG1 antibodies for further evaluation. The light and heavy chain variable region sequences of these antibodies are reported in Figures 11A and B.

The fifteen redesigned Abs were tested for their ability to block PD-1 binding to 293 cells expressing human or murine PD-L1 by electrochemiluminescence (ECL) cell binding assay. (Table 1 - bottom half: In Table 1, "Format 1" describes the binding of soluble human PD-1-Fc to human PD-L1-transfected 293 cells; "Format 2" describes the binding of murine PD-1-Fc to murine PD-L1-transfected 293 cells, and "Format 3" describes the binding of human PD-1 to murine PD-L1-transfected 293 cells.) Although all fifteen avidity-improved Abs achieved significant cross-reactivity against murine PD-L1, YW243.55S70 was selected as the lead candidate to proceed based on its ability to block both human and murine PD-L1 binding to PD-1 (Table 1: _IC50 values of 49 pM and 22 pM, respectively).

Table 1

Example 2

Characterization of anti-PD-L1 antibodies (BIAcore)

The binding affinity of anti-PD-L1 phage antibodies YW243.55 and YW243.55S70 against recombinant human and murine PD-L1 was measured by surface plasmon resonance (SRP) using a BIAcore ^™ -3000 instrument. Recombinant human PD-L1-Fc (R&D Systems, cat#156-B7) and recombinant murine PD-L1-Fc (R&D Systems, cat#1019-B7) were directly coated onto a CM5 biosensor chip to obtain approximately 500 response units (RU). For kinetic measurements, two-fold serial dilutions (3.9 to 500 nM) were injected into PBT buffer (PBS containing 0.05% Tween-20) at 25°C at a flow rate of 30 μL/min. Association rates ( _kon ) and dissociation rates ( _koff ) were calculated using a simple one-to-one Languir binding model (BIAcore Evaluation Software version 3.2). The equilibrium dissociation constant (kD) was calculated as the ratio _koff / _kon .

The measured binding affinities of anti-PD-L1 phage antibody clones YW243.55 and YW243.55.S70 are reported in Table 2 below.

Table 2

BIAcore binding affinity

Example 3A

Specificity of anti-PD-L1 Abs for human, rhesus, and mouse PD-L1 – FACS and radioligand cell binding assays

This example demonstrates the specificity of the anti-PD-L1 antibodies of the present invention for human, rhesus monkey, and mouse PD-L1. Furthermore, this example demonstrates the affinity of the antibodies for mouse and human PD-L1 expressed on the cell membrane of 293-transfected cells.

Human and murine PD-L1 were stably transfected into 293 cells. Cells were harvested and plated at 150,000 cells per well in 96-well plates for binding studies.

Rhesus monkey blood was obtained from the Southwest Foundation for Biomedical Research (San Antonio, Texas). The blood was diluted with an equal volume of PBS and covered on 96% Ficoll-Paque (GE Healthcare) for isolation of monocytes. Monocytes were lysed from erythrocytes using red blood cell lysis buffer (Qiagen) and cultured overnight in 6-well plates at 1.5 x ¹⁰ cells/ml with 5 ng/ml PMA plus 1 μM ionomycin. The culture medium was RPMI1640 containing 10% fetal bovine serum, 20 μM HEPES, and the following additives from Gibco at a 1:100 dilution: Gluta-MAX, sodium pyruvate, penicillin/streptomycin, and non-essential amino acids. The next day, cells were harvested and aliquoted into 96-well plates for binding studies (approximately 120,000 cells per well).

PD-L1 antibody YW243.55.S70 or an antibody control was titrated starting at 10 μg/ml in three-fold serial dilutions and bound to cells in a 50 μl volume on ice for 25 minutes. Cells were washed and then bound to anti-human IgG PE (Caltag) at 20 μg/ml on ice for 25 minutes. Rhesus macaque cells were also co-stained with CD3 FITC and CD4 APC (BD Biosciences) to distinguish CD4+ T cells.

All samples were run on a Beckman Dickinson FACSCalibur and Tree Star, Inc. software was used to analyze the mean fluorescence intensity of PD-L1 binding data as a function of anti-PD-L1 antibody concentration; EC ₅₀ values (Ab concentration associated with half-maximal binding) were calculated using Kaleidagraph. In addition, equilibrium binding studies were performed to define the precise affinity (Kds) of YW24355S70 for binding to human and mouse PD-L1 expressed on 293 cells (Example 3B). These values are summarized in Table 3 below:

Table 3

EC ₅₀ Summary

Example 3B

Measurement of Anti-PD-L1 Ab Avidity for Human and Murine PD-L1 – Equilibrium Binding Radioligand Cell Binding Assay

293 cells transfected with human or mouse PD-L1 were cultured at 37°C in 5% _CO2 in growth medium consisting of RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 1X penicillin-streptomycin. Cells were washed with binding buffer (50:50 DMEM/F12 with 2% FBS and 50 mM Hepes, pH 7.2) and plated into 96-well plates at approximately 230,000 cells per well in 0.2 mL of binding buffer. The anti-PD-L1 antibody, YW243.55.S70.hIgG, was iodinated using the iodogen method. The radiolabeled anti-PD-L1 antibody was purified from free ¹²⁵ I-NA by gel filtration using a NAP-5 column; the purified Ab had a specific activity of 17.41 μCi/μg. A 50 μL volume of competition reaction mixture containing a fixed concentration of iodinated antibody and a serial dilution of unlabeled antibody at a decreasing concentration was placed in a 96-well plate. 293 stably transfected cell lines expressing human PD-L1 and mouse PD-L1 were cultured in growth medium at 37°C in 5% CO ₂ , consisting of 50:50 DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine, 1X penicillin-streptomycin. Cells were washed with binding buffer (50:50 DMEM/F12 with 2% FBS, 50mM HEPES, pH 7.2, and 2mM sodium azide) and added to 50 μL of competition reaction mixture at a density of approximately 200,000 cells in 0.2 mL of binding buffer. The final concentration of the iodinated antibody in each competition reaction with cells was 150 pM (120,000 cpms/0.25 mL) and the final concentration of the unlabeled antibody in the competition reaction with cells was varied, starting at 500 nM and then decreasing 2-fold (for 10 concentrations). The competition reaction with cells was incubated for 2 hours at room temperature. For each concentration of unlabeled antibody, the competition reaction with cells was repeated three times for determination. After 2 hours of incubation, the competition reaction was transferred to a Millipore Multiscreen filter plate and washed 4 times with binding buffer to separate free antibody from the bound iodinated antibody. The filter was counted on a Wallac Wizard 1470 gamma counter (PerkinElmer Life and Analytical Sciences Inc. Wellesley, MA). The binding data were evaluated using NewLigand software (Genentech), which uses the fitting algorithm of Munson and Robard to determine the binding affinity of the antibody. Musson et al., Anal. Biochem. 107 : 220-39 (1980).

As shown in Table 3, Kd values as determined by Scatchard analysis confirm the EC50 values of anti-PD-L1 antibodies that bind to human and murine PD-L1.

Example 4

Selectivity and affinity (IC ₅₀ ) of anti-PD-L1 Ab

This example demonstrates the use of binding selectivity and affinity (eg, IC ₅₀ ) assays to evaluate the ability of full-length anti-PD-L1 antibodies of the invention to block PD-L1 binding to both PD-1 and B7.1.

method:

hB7.1-Fc-Biotin and hPD-1-Fc-Biotin combined with hPD-L1-Fc ELISA (Format 4)

Nunc Maxisorp 384-well plates were coated overnight with 25 μL of 250 ng/mL hPD-L1-Fc in PBS. The wells were washed three times with PBS with 0.05% Tween (wash buffer) on a microplate washer and blocked with PBS with 0.5% BSA. 12.5 μL of 2X concentration of antibody in PBS with 0.05% Tween, 0.5% BSA (assay diluent) was added to each well. Then, 12.5 μL of 250 ng/mL (2X concentration) of hB7.1-Fc-biotin in assay diluent was added and the plate was incubated with shaking for 1.5 hours. The wells were washed six times with wash buffer and 25 μL of streptavidin-HRP (1:40,000 in assay diluent, GE Healthcare) was added. The plate was incubated with agitation for 30 minutes and the wells were washed six times with wash buffer. 25 μL of TMB substrate (Kirkegaard and Perry laboratories) was added for one hour and the reaction was stopped with 25 μL of 1 M phosphoric acid. The absorbance was read at 450 nm and _IC50 values were analyzed as described in Example 1 for the ECL-cell binding assay.

Forms 5, 6, and 7:

For hPD-L1-Fc-binding hPD-1-Fc-biotin (Format 5), the format was similar to the above assay, except that hPD-1-Fc-biotin was used for binding instead of hB7.1-Fc-biotin. The TMB substrate reaction time was 17 minutes.

For mB7.1-Fc-biotin conjugated to mPD-L1-Fc (Format 6), this format was similar to Format 5, except that mPD-L1-Fc was used for coating the plate instead of hPD-L1-Fc, and mB7.1-Fc-biotin was used for conjugation instead of hB7.1-Fc-biotin. The TMB substrate reaction time was 7 minutes.

For mPD-L1-Fc binding to mPD-1-Fc-biotin (Format 7), the format was similar to the mouse ELISA mentioned above, except that mPD-1-Fc-biotin was used for binding instead of mB7.1-Fc-biotin. The TMB substrate reaction time was 5 minutes.

result:

Table 4 reports the IC ₅₀ evaluation of the affinity-matured phage anti-PD-L1 antibody YW243.55.S70 for blocking the interaction between the indicated binding pairs. YW243.55S70 was able to block human PD-L1 binding to hB7.1Fc at a half-maximal inhibitory concentration of 38 pM, which is relatively equivalent to its IC ₅₀ value for blocking the PD-L1/PD-1 interaction (42 pM). Biacore studies, which measured the ability of YW243.55S70 to block the interaction of PD-L1 with both PD-1 and B7.1, were consistent with these ELISA results (data not shown).

Table 4

Example 5

PMEL/B16 in vitro assay using anti-PD-L1 antibody YW243.55.S70 to enhance CD4+ and CD8+ T cell activity Certainly

This example shows the effect of the anti-PD-L1 antibody of the present invention on the activation of CD8 ⁺ T cells of PMEL T cell receptor transgenes, as measured by enhancing the production of γ-IFN in response to the melanocyte peptide gp100. In this procedure, CD8+T cells are obtained from PMEL TCR transgenic mice whose CD8+T cells express TCR specific for the gp100 peptide. After purifying the CD8+T cells, multiple rounds of stimulation are performed to produce and amplify activated CD8+T cells, which will then in turn upregulate PD-1 expression. In parallel, B16 melanoma cells are treated with IFN-γ to upregulate their PD-L1 expression. The cells are then co-cultured in the presence of anti-PD-L1 antibodies and the effect on IFN-γ production is evaluated. B16 cells are selected for the third stimulation because they endogenously express gp100 peptides at low levels (as opposed to exogenously applied peptides). Furthermore, because these cells also do not express PD-L2, B7.1, or B7.2, the impact of additional signaling unrelated to PD-L1 (e.g., signaling through CD28 or CTLA-4 or PD-L2-induced signaling through PD-1) can be minimized.

PMEL assay:

As shown in FIG3 , anti-PD-L1 antibodies enhanced the percentage of IFN-γ-producing PMEL CD8 ⁺ T cells and the mean levels of IFN-γ produced in response to the indicated amounts of gp100 peptide.

D.011.10 In vitro assays:

Similar assays using Ova-specific TCR Tg CD4+ T cells showed that prior stimulation with Ova peptide to induce PD-1 expression enhanced T cell proliferation in the presence of anti-PD-L1 Ab (Figure 4). In the final stimulation, irradiated A20B cells expressing PD-L1 were used to present the indicated concentrations of Ova peptide to DO.11.10 T cells. Notably, the contribution of the PD-1/PD-L1 axis was more pronounced at lower levels of antigen receptor stimulation, a level that more closely reflects the physiologically relevant intensity of stimulation.

Materials and methods:

PMEL assay

Initial stimulation (days 0-4)

Spleens and mesenteric lymph nodes were harvested from PMEL transgenic T cell recipient mice. Organs were crushed into a single-cell suspension and red blood cells were lysed. CD8 ⁺ T cells were isolated using a CD8 ⁺ T cell isolation kit and an AutoMACS cell separator (Miltenyi Biotec) according to the manufacturer's instructions.

Spleens were isolated from non-transgenic, sex-matched mice and crushed into a single-cell suspension, and erythrocytes were lysed. Cells were sensitized with 0.1 μg/ml gp100 peptide at 37°C for two hours and washed.

Cells were co-cultured for 4 days in 96-well flat-bottom plates with 200,000 PMEL CD8 ⁺ T cells and 75,000 gp100-sensitized splenocytes. The culture medium consisted of Iscove's modified Dulbecco's medium plus 10% fetal bovine serum and 20 μM HEPES, with the following supplements from Gibco diluted 1:100: Gluta-MAX, sodium pyruvate, penicillin/streptomycin, and non-essential amino acids.

Second stimulation (Day 4-7)

The PMEL cultures were spun down and the culture medium was aspirated with a multichannel pipette. Fresh culture medium was added and mixed to wash the cells, then spun again. Most of the culture medium was removed and the antibody (YW243.55.S70 or none) was added at a final concentration of 10 μg/ml. Conditions were set in replicate wells so that average IFN-γ production could be evaluated at the endpoint.

DC-1 cells were pulsed with 0.1 μg/ml gp100 peptide at 37°C for 2 hours and washed. Gp100-pulsed DC-1 cells were added at 40,000 cells/well to wash PMEL cultures. PMEL and DC-1 + antibody were co-cultured for 3 days.

Third stimulation (Day 7-8)

On day 6 (one day before the third stimulation), B16 melanoma cells were incubated with 20 ng/ml of murine IFN-γ (R&D Systems) overnight to upregulate their PD-L1 expression.

On day 7, the PMEL cultures were spun down and the medium was aspirated using a multichannel pipette. Fresh medium was added and mixed, followed by another spin. Most of the medium was removed and the antibody was added at a final concentration of 10 μg/ml.

After overnight stimulation with IFN-γ, B16 cells were washed and divided into three groups for incubation with no gp100, 1 ng/ml gp100 (gp100 low), or 10 ng/ml gp100 (gp100 high) for two hours. The cells were washed and then added to the washed PMEL+Ab cultures at 40,000 cells per well and incubated overnight.

Intracellular staining of IFN-γ on day 8

Golgi-Plug (BD Biosciences) was added in the last 5 hours of culture according to the manufacturer's instructions. IFN-γ intracellular staining was performed using the BD Biosciences Cytofix/CytopermFixation/Permeabilization solution kit, and all staining antibodies were also from BD Biosciences, according to the manufacturer's instructions. Cells were surface stained with CD8a PE and Thy1.1 FITC and intracellularly stained with IFN-γ APC at saturating concentrations.

All samples were run on a Beckman Dickinson FACSCalibur and data were analyzed using Tree Star, Inc. FLOWJO ^™ software.

D011.10 In vitro assay

Spleens and mesenteric lymph nodes from DO11.10 transgenic mice were harvested, crushed into a single-cell suspension, and red blood cells were lysed. Cells were cultured in 6-well plates at a density of 1 x ¹⁰ cells/mL with 0.3 μM Ova peptide for 72 hours. The culture medium consisted of RPMI 1640 supplemented with 10% fetal bovine serum, 20 μM HEPES, and the following supplements from Gibco at a 1:100 dilution: Gluta-MAX, sodium pyruvate, penicillin/streptomycin, and non-essential amino acids.

After the initial stimulation, cells were harvested and CD4 ⁺ T cells were purified using a mouse CD4 T cell purification kit according to the manufacturer's instructions (Miltenyi Biotec). The purified CD4 ⁺ T cells were then allowed to rest overnight.

The next day, cells were harvested, washed, and co-cultured with irradiated (10,000 rads) A20 cells. Co-cultures were set up in triplicate wells of a 96-well U-bottom plate with 50,000 CD4 ⁺ T cells to 40,000 A20 cells, titrated with Ova peptide, and a final concentration of 20 μg/ml of antibody. After 48 hours, cultures were primed overnight with 1 μCi/well of 3H-thymidine and frozen the next day. Plates were then thawed, harvested using a cell harvester, and read in a beta-counter.

Example 6

Enhancement of human CD8+ T cell proliferation by anti-PD-L1 in mixed lymphocyte reaction

Figure 5 demonstrates that anti-PD-L1 (e.g., YW243.55.S1) enhances the proliferation of human CD8 T cells in response to MHC- mismatched donor cells. According to the instructions of each manufacturer, CD8+ T cells (StemCell Technologies) were first used to enrich the CD8+ T cells of the response from the whole blood of donor A. The cells were then diluted with an equal volume of phosphate-buffered saline (PBS) and separated by gradient centrifugation by covering them on Ficoll-Paque Plus (GE Healthcare). After separation, the cells were stained with CD8APC (BD Biosciences) and were found to be 78% CD8+ T cells. The cells were fluorescently labeled with 2.5 μM CFSE tracer dye (Molecular Probes).

In order to be used as allogeneic antigen presenting cells (APC), first separate mononuclear cells from the whole blood of donor B, then exclude CD3+ T cells. Dilute blood with equal volume of PBS and separate mononuclear cells after gradient centrifugation on Ficoll. Cells are stained and washed with CD3FITC (BD Biosciences), then incubated with anti-FITC glass beads (Miltenyi Biotec). Then, on AutoMACS cell separator (Miltenyi Biotec), exclude CD3FITC positive cells. Then cells are irradiated 2500rads on cesium irradiator.

Cells were co-cultured with 150,000 CD8+ T cells and 150,000 APCs in 96-well flat-bottom plates with 10 μg/ml of antibody for 5 days. The culture medium consisted of RPMI 1640 supplemented with 10% fetal bovine serum, 20 μM HEPES, and the following supplements from Gibco diluted 1:100: Gluta-MAX, sodium pyruvate, penicillin/streptomycin, and non-essential amino acids.

On day 5, cells were harvested, washed, and stained with CD8-biotin followed by streptavidin-PerCp (BD Biosciences). Samples were run on a Beckman Dickinson FACSCalibur and data were analyzed using Tree Star, Inc. FlowJo software.

In the presence of anti-PD-L1, an approximately 45% enhancement in the proliferation of CD8 T cells responding to cells from MHC-mismatched donors was observed.

Example 7

Effects of PD-L1 Blockade of LCMV in an In Vivo Model

Under conditions of chronic stimulation, T cells have been shown to upregulate and maintain expression of the inhibitory receptor PD-1. Ligation of PD-1 through one of its two ligands, PD-L1 and PD-L2, contributes to the refractory state of chronically activated T cells, weakening their response to their cognate antigens. In mice persistently infected with lymphocytic choriomeningitis virus (LCMV), blockade of PD-1 or its ligand PD-L1 is sufficient to resurrect chronically refractory T cells and enhance the intensity and functional quality of anti-viral T cell responses. Similarly, humans chronically infected with HIV or HCV display T cells that are refractory to stimulation, and the activity of these T cells can be enhanced in vitro by blocking PD-1 or PD-L1. Therefore, the activity of PD-L1 blockade in the LCMV model suggests therapeutic potential for enhancing anti-viral and anti-tumor immunity.

For in vivo experiments with LCMV in mice, we have redesigned a humanized anti-PD-L1 antibody (YW243.55S70) by cloning phage-derived heavy and light chain variable region sequences upstream of mouse IgG2a heavy chain and mouse kappa light chain constant domains. To prevent antibody-mediated cytotoxicity of cells expressing PD-L1, positions 265 (aspartic acid) and 297 (asparagine) were changed to alanine (DANA) by inhibiting Fcγ receptor binding. Shields, RL et al. J. Biol Chem 2001 276 (9): 6591-6604. To test the ability of the anti-PD-L1 antibody to enhance anti-viral immunity in chronic infection, mice were infected with 2 x 10 ⁶ plaque-forming units (pfu) of clone 13 LCMV on day 0 or the Armstrong strain of LCMV as a reference control. The schematic experimental design is presented in Figure 6. Infection with clone 13 resulted in chronic infection, characterized by T cell expansion but ineffective viral clearance, whereas Armstrong LCMV was cleared within 8-10 days of infection. On day 14, mice were treated with anti-PD-L1 or control mIgG delivered at a dose of 10 mg/kg 3x/week. CD8 T cell function and viral titers in blood and tissues were analyzed on days 21 and 28.

Consistent with the data published by Barber et al., Nature 439 : 682-7 (2006), this example shows that anti-PD-L1 Ab enhances the ability of cytotoxic lymphocytes that respond to LCMV after a 2-week treatment regimen in chronic LCMV infection. Figure 7A shows the percentage of CD8 T cells that respond to gp33LCMV-specific peptides, which express CD107a on their cell surface. Plasma membrane expression of CD107a (which is generally expressed intracellularly) accompanies the degranulation process and is therefore used as a surrogate marker for degranulation. Relative to the response of cells from acute Armstrong LCMV infection, cells from animals infected with the chronic strain (clone 13) are impaired in degranulation (control Ig group), while PD-L1 blockade can reconstruct CD8+ degranulation to a level that is comparable to those observed in Armstrong infection. Similarly, 7B demonstrates that the % of CD8 T cells producing IFN-γ in response to LCMV gp33 was increased in the anti-PD-L1-treated group relative to control Ig.

Then, the effect of anti-PD-L1Ab on reducing or eradicating LCMV virus in blood and tissues was tested. In Figure 8A, the figure shows the logarithmic viral titers in the tissues shown in control Ig and PD-L1 treated animals on the 21st and 28th days after infection with clone 13LCMV. Antibody treatment began on the 14th day after infection. Blocking PD-L1 resulted in a very significant reduction in viral titers in blood, liver, brain, lungs and kidneys. Impressively, in 3 of 5 mice, α-PD-L1Ab reduced blood LCMV titers to levels below detection (<1x ^10-5 ). In a subsequent comparative design experiment, viral eradication in blood and liver was observed in 5/5 mice treated with 10mg/kg or 2mg/kg 3x/weekly doses of anti-PD-L1 for 2 weeks (data not shown). The lower figure shows the kinetics of viral titer reduction in blood and indicates that the viral titer in the anti-PD-L1 treated group was reduced by an average of 96.8% relative to the control on day 28. These data support the importance of the PD-1/PD-L1 pathway in suppressing T-cell responses in chronic infection and are consistent with the in vitro effects of PD-L1 blockade on T cells obtained from humans with chronic infections such as hepatitis C and HIV.

Materials and methods:

Determination of % IFN-γ produced by CD8 T cells in response to LCMV gp33 peptide

Spleens were isolated from infected mice and single cell suspensions were generated by crushing the organs in complete medium containing 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin/streptomycin, and 10 mM 2-mercaptoethanol in IMDM (Invitrogen Inc., Carlsbad, CA). Red blood cells were lysed using ACK lysis buffer (0.15 M _NH4Cl , 10 mM _KHCO3 , 0.1 mM EDTA). To measure antigen-specific CD8 T cell responses, splenocytes were washed in complete medium and restimulated in vitro with the LCMV peptide GP33 (KAVYNFATC, ProImmune Inc., Bradenton, FL) for 4 hours. 1 x 10 6 splenocytes were cultured in 96-well flat-bottom plates in the presence of 100 units/ml human interleukin-2 (Sigma-Aldrich, St. Louis, MO), 1 μl/ml brefeldin A, and 1 μl/ml ( ¹ :1000 dilution) monensin (BD pharmingen) and anti-CD107a FITC (clone ID4B, BD Biosciences, San Jose, CA) with 100 ng/ml GP33 peptide. After incubation, cells were washed once in PBS containing 2% fetal bovine serum and stained for cell surface markers using the following fluorochrome-conjugated antibodies: anti-CD8 APC (clone 53.67, BD Biosciences, San Jose, CA), anti-CD4 PerCp-Cy5.5 (clone RM4-5, BD Biosciences, San Jose, CA), and anti-PD-1 PE (clone J43, BD Biosciences, San Jose, CA). Intracellular IFN-γ staining was performed using Cytofix Cytoperm Plus kit (BD Biosciences, San Jose, CA) according to the manufacturer's instructions. Anti-IFN-γ PE-Cy7 (clone XMG1.2, eBioscience Inc. San Diego, CA) was used. To detect the number of GP33-specific CD8 T cells, fresh splenocytes were stained with GP33 pentamers (H2-Db attached to APC, ProImmune Inc., Bradenton, FL) according to the manufacturer's instructions. Data were collected using BD FACSAria (BD Biosciences, San Jose, CA) and analyzed using FlowJo Software (Tree Star Inc. Ashland OR).

Determine LCMV virus titer:

MC57 fibrosarcoma cells were infected with 10-fold serial dilutions of blood or tissue homogenate containing LCMV in complete IMDM. The reaction was then incubated at 37°C in a tissue culture incubator for 2-6 hours and then covered with DMEM containing 1% methylcellulose. After that, the cells were incubated for 3-5 days and then the methylcellulose layer was removed by aspiration. The cells were fixed with PBS/4% paraformaldehyde, permeabilized with 0.5% Triton-x for 20 minutes, washed in PBS, and then blocked in 10% FCS for 1 hour under gentle shaking. LCMV was stained with VL4 antibody (1 hour), washed 2 times, and then developed with anti-rat HRP (1:400) in blocking buffer. After that, the cells were washed 3 times and then o-phenylenediamine substrate (SIGMA P8806-50TAB 3mg/piece) was added to the wells to develop the color.

Example 8

PD-L1 blockade in cancer

It is now clear that many tumors utilize expression of PD-1 ligands as a means of attenuating anti-tumor T cell responses. Several human cancers have been characterized by elevated expression of PD-L1 in both tumors and tumor-infiltrating leukocytes, and this elevated PD-L1 expression is often associated with a worse prognosis. Mouse tumor models have demonstrated similar increases in intratumoral PD-L1 expression and suggest a role for the PD-1/PD-L1 pathway in suppressing tumor immunity.

Here, we present experiments demonstrating the effect of PD-L1 blockade on orthotopic tumor growth of MC38.Ova murine colorectal cancer cells in syngeneic C57B6 mice (Figure 9A). These cells, which express ovalbumin on their cell surface via retroviral transduction, express PD-L1 but not PD-L2, as assessed by flow cytometry (bar graph – Figure 9B). Mice were inoculated subcutaneously with 500,000 MC38.Ova cells on day 0. During the study, 10 mice/group were treated 3 times weekly with 10 mg/kg of anti-PD-L1 (YW243.55S70-mouse IgG2a-DANA), control Ig, or a blocking anti-CTLA4 Ab (UC10-4F10-11) on day 1 or day 14 (when tumors had reached an average size of 250 ^mm³ ). PD-L1 blockade, whether as a single agent or in late intervention, was highly effective in preventing tumor growth. In contrast, blocking another inhibitory molecule expressed on T cells (CTLA4) showed no evidence of inhibition of tumor growth. These results suggest a unique role for the PD-1/PD-L1 axis versus CTLA4/B7 in suppressing anti-tumor immune responses and support the therapeutic potential of antibodies that block the interaction of PD-L1 with PD-1 and B7.1 for human cancers.

MC38.Ova syngeneic tumor model: Methods. On day 0, 70 animals were subcutaneously inoculated with 500,000 MC38.Ova cells in 100 microliters of HBSS + Matrigel. Starting at D1, 20 mice were added to one of the two treatment groups (see below: Group 1 or Group 2). The remaining 40 mice were allowed to grow tumors until day 14. Of these 40 mice, 30 mice with similar sized tumors were added to one of the three treatment groups (Groups 3-5). Tumors were measured and mice were weighed 2x/week. Mice euthanized due to dissimilar tumor volumes were not added to the following treatment groups:

Group 1: anti-gp120 antibody, 10 mg/kg IP, 100 μL, D1, 3x/week

Group 2: anti-PD-L1 antibody, 10 mg/kg IP, 100 μL, D1, 3x/week

Group 3: anti-gp120 antibody, 10 mg/kg IP, 100 μL, D14, 3x/week

Group 4: anti-PD-L1 antibody, 10 mg/kg IP, 100 μL, D14, 3x/week

Group 5: anti-CTLA-4 antibody, 10 mg/kg IP, 100 μL, D14, 3x/week

***Groups 1 and 2 started dosing on D1; Groups 3, 4, and 5 started dosing on D14.

Example 9

Combining Anti-PD-L1 with Other Agents to Provide Anti-Tumor Effects or Immune Enhancement – MC38.Ova Model

At day 0, 150 animals were inoculated subcutaneously with 500,000 MC38.Ova cells in 100 microliters of HBSS+ matrigel. The mice were allowed to grow tumors. 2x/week mice were weighed and measured until the 11th day (when the tumor volume was 100-200 mm ³ between). At day 11, after tumor measurement, mice were added to one of the following 12 treatment groups. Due to dissimilar tumor volumes, mice euthanized were not added to the following treatment group. At day 12, gemcitabine (group 4) was started to process, but at day 14, the remaining antibody groups were started to process. All volumes in the inert carrier were 100 μl, and other details are as reported below:

Group 1: anti-gp120 antibody, 10 mg/kg IP, 100 μL, 3x/week x 5, n=10

Group 2: anti-PD-L1 antibody, 10 mg/kg IP, 100 μL, 3x/week x 5, n=10

Group 3: anti-VEGF antibody, 5 mg/kg IP, 100 μL, 2x/week x 5, n=10

Group 4: gemcitabine, 40 mg/kg IP, 100 μl, Day 12, 16, 20, n=10

Group 5: anti-PD-L1 antibody + anti-gp120 antibody, n=10

Group 6: anti-PD-L1 antibody + anti-VEGF antibody, n=10

Group 7: anti-PD-L1 antibody + gemcitabine, n = 10

Group 8: anti-gp120 antibody + gemcitabine, n = 10

Group 9: anti-gp120 antibody + anti-VEGF, n=10

Day 12: Mice from Group 1 were anesthetized and blood (100 μl) was collected from the retrobulbar vein for CBC analysis.

Day 14 and Day 22: Mice from Group 4 were anesthetized and blood (100 μl) was collected from the retrobulbar vein for CBC analysis.

Day 19: All mice except mice from Group 4 were anesthetized and blood (100 μl) was collected from the retrobulbar vein for CBC analysis.

Day 26: All mice except mice from Group 4 were anesthetized and blood (100 μl) was collected from the retrobulbar vein for PK analysis.

Tumors were measured and mice were weighed 2X/week. Animals showing >15% weight loss were weighed daily and euthanized if they lost >20% of their body weight. Mice were euthanized when tumor volume exceeded 3,000 mm ³ or after 3 months if no tumors had formed.

This study showed (Figure 10) that PD-L1 blockade was more effective than the induction regimen of α-VEGF and gemcitabine alone.

Example 10

Expression of anti-PD-L1 antibodies in mammalian cells

This example illustrates the preparation of potentially glycosylated forms of anti-PD-L1 antibodies by recombinant expression in mammalian cells.

Vector pRK5 (see EP 307,247 published March 15, 1989) is used as an expression vector. Optionally, DNA encoding the light and/or heavy chains of the antibody is ligated into pRK5 after digestion with selected restriction enzymes to allow insertion of the DNA, using a ligation method such as that described in Sambrook et al. (supra).

In one embodiment, the selected host cell can be a 293 cell. Human 293 cells (ATCC CCL 1573) are cultured in a tissue culture dish in a medium such as DMEM supplemented with fetal bovine serum and optional nutrients and/or antibiotics until confluent. Approximately 10 μg of DNA (if the DNA encodes pRK5-antibody) is mixed with approximately 1 μg of DNA encoding the VA RNA gene [Thimmappaya et al., Cell, 31 : 543 (1982)] and dissolved in 500 μL of 1 mM Tris-HCl, 0.1 mM EDTA, and 0.227 _M CaCl₂. To this mixture, 500 μL of 50 mM HEPES (pH 7.35), 280 mM NaCl, and 1.5 mM _NaPO₄ are added dropwise, and a precipitate is formed at 25°C for 10 minutes. The precipitate is suspended and added to the 293 cells and allowed to stand at 37°C for approximately 4 hours. The culture medium was aspirated and 2 ml of PBS containing 20% glycerol was added for 30 seconds. The 293 cells were then washed with serum-free medium, fresh medium was added, and the cells were incubated for approximately 5 days.

Approximately 24 hours after transfection, the culture medium is removed and replaced with culture medium alone or with culture medium containing 200 μCi/ml ³⁵ S-cysteine and 200 μCi/ml ³⁵ S-methionine. After a 12-hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel. The treated gel is dried and exposed to film for a selected period of time to visualize the presence of the antibody. Cultures containing transfected cells can be further incubated in serum-free medium, and the culture medium tested in selected bioassays.

In an alternative technique, the antibody can be transiently introduced into 293 cells using the dextran sulfate method described by Somparyrac et al., Proc. Natl. Acad. Sci., 12 : 7575 (1981). 293 cells are cultured to maximum density in a rotary shaker and 700 μg of DNA encoding pRK5-antibody is added. Cells are first concentrated from the rotary shaker by centrifugation and washed with PBS. The DNA-dextran precipitate is incubated on the cell pellet for 4 hours. The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and placed back into a rotary shaker containing tissue culture medium, 5 μg/ml bovine insulin, and 0.1 μg/ml bovine transferrin. After approximately 4 days, the conditioned medium is centrifuged and filtered to remove cells and debris. The sample containing the expressed antibody can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography.

In another embodiment, the antibody can be expressed in CHO cells. The DNA encoding the antibody linked to pRK5 can be transfected into CHO cells using known reagents such as CaPO ₄ or DEAE-dextran. As described above, the cell culture can be incubated and the culture medium replaced with culture medium (alone) or with culture medium containing a radioactive marker such as ³⁵ S-methionine. After determining the presence of the antibody, the culture medium can be replaced with serum-free medium. Preferably, the culture is incubated for about 6 days and the conditioned medium is then harvested. The culture fluid containing the expressed antibody can then be concentrated and purified by any selected method.

Variants of epitope-tagged antibodies can also be expressed in host CHO cells. The DNA encoding the antibody ligated into pRK5 can be subcloned outside the pRK5 vector. The subcloned insert can be PCR-fused into a baculovirus expression vector in the same reading frame as the selected epitope tag (such as a polyhistidine tag). The polyhistidine-tagged DNA insert encoding the antibody can then be subcloned into an SV40-driven vector containing a selection marker (such as DHFR) to select stable clones. Finally, CHO cells can be transfected with the SV40-driven vector (as described above). Labeling can be performed as described above to verify expression. The culture medium containing the expressed poly-His-tagged antibody can then be concentrated and purified by any selected method, such as Ni ²⁺ -chelate affinity chromatography.

The antibodies may also be expressed in CHO and/or COS cells by transient expression protocols, or in CHO cells by other stable expression procedures.

Stable expression in CHO cells was performed using the following procedure: The proteins were expressed as IgG constructs (immunoadhesins) in which the coding sequence for the soluble form of the respective protein (e.g., the extracellular domain) was fused to IgG1 constant region sequences including the hinge, CH2, and CH3 domains, and/or in a poly-His-tagged format.

After PCR amplification, each DNA was subcloned into a CHO expression vector using standard techniques as described in Ausubel et al., Current Protocols of Molecular Biology, Unit 3.16, John Wiley and Sons (1997). CHO expression vectors were constructed with compatible restriction sites at the 5' and 3' ends of the DNA of interest to allow for convenient shuttling of the cDNA. Vectors for expression in CHO cells are described in Lucas et al., Nucl. Acids Res. 24 :9 (1774-1779 (1996) using the SV40 early promoter/enhancer to drive expression of the cDNA of interest and dihydrofolate reductase (DHFR). DHFR expression allows selection for stable maintenance of the plasmid after transfection.

Using commercial transfection reagents (Quiagen) or (Boehringer Mannheim), 12 μg of the desired plasmid DNA was introduced into approximately 10 million CHO cells. The cells were cultured as described in Lucas et al. (supra). Approximately 3 x 10 ⁻⁷ cells were frozen in ampoules for further culture and production as described below.

Thaw the ampoule containing the plasmid DNA in a water bath and shake to mix. Transfer the contents to a centrifuge tube containing 10 mL of culture medium and centrifuge at 1000 rpm for 5 minutes. Aspirate the supernatant and resuspend the cells in 10 mL of selective culture medium (0.2 μm filtered PS20 containing 5% 0.2 μm diafiltered fetal bovine serum). Then, aliquot the cells into 100 mL spinner flasks containing 90 mL of selective culture medium. After 1-2 days, transfer the cells to a 250 mL spinner flask containing 150 mL of selective growth medium and incubate at 37°C. After another 2-3 days, inoculate 250 mL, 500 mL, and 2000 mL spinner flasks at 3 x ¹⁰ cells/mL. Replace the cell culture medium with fresh culture medium by centrifugation and resuspending in production culture medium. While any suitable CHO culture medium can be used, the production culture medium described in U.S. Patent No. 5,122,469, issued June 16, 1992, can be used. 3L production roller bottles were inoculated with 1.2x ¹⁰⁶ cells/mL. On day 0, cell number and pH were determined. On day 1, the roller bottles were sampled and filtered air was started. On day 2, the roller bottles were sampled, the temperature was changed to 33°C, and 30mL 500g/L glucose and 0.6mL 10% antifoam (e.g., 35% polydimethylsiloxane emulsion, Dow Corning 365 medical emulsion) were added. Throughout the production process, the pH was adjusted to remain at about 7.2 when necessary. After 10 days, or until viability dropped to below 70%, the cell culture was harvested by centrifugation and filtered through a 0.22 μm filter membrane. The filtrate was stored at 4°C or immediately loaded onto the column for purification.

For poly-His-tagged constructs, protein was purified using a Ni-NTA column (Qiagen). Prior to purification, imidazole was added to the conditioned medium to a concentration of 5 mM. The conditioned medium was pumped at a flow rate of 4-5 ml/min onto a 6 ml Ni-NTA column equilibrated at 4°C in 20 mM Hepes buffer (pH 7.4) containing 0.3 M NaCl and 5 mM imidazole. After loading, the column was washed with additional equilibration buffer, and the protein was eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein was then desalted using a 25 ml G25 Superfine column (Pharmacia), stored in a storage buffer containing 10 mM Hepes, 0.14 M NaCl, and 4% mannitol (pH 6.8), and stored at -80°C.

Immunoadhesin (containing Fc) constructs were purified from conditioned medium as follows. Conditioned medium was applied to a 5 ml protein A column (Pharmacia) equilibrated in 20 mM sodium phosphate buffer (pH 6.8) using a pump. After loading, the column was thoroughly washed with equilibration buffer and then eluted with 100 mM citric acid (pH 3.5). The eluted protein was immediately neutralized by collecting 1 ml fractions into a tube containing 275 μL 1 M Tris buffer (pH 9). The highly purified protein was then desalted as described above for the poly-His-tagged protein and stored in storage buffer. Homogeneity was assessed by SDS polyacrylamide gel and N-terminal amino acid sequencing following Edman degradation.

Example 11

Expression of anti-PD-L1 antibodies in Escherichia coli

This example illustrates the preparation of a non-glycosylated form of an anti-PD-L1 antibody by recombinant expression in E. coli.

First, the DNA sequence encoding the anti-PD-L1 antibody is amplified using selected PCR primers. The primers should contain restriction enzyme sites corresponding to the restriction enzyme sites on the selected expression vector. A variety of expression vectors can be used. An example of a suitable vector is pBR322 (derived from Escherichia coli; see Bolivar et al., Gene, 2 : 95 (1977)), which contains genes for ampicillin and tetracycline resistance. The vector is digested with restriction enzymes and dephosphorylated. The PCR amplified sequence is then ligated into the vector. The vector preferably includes sequences encoding an antibiotic resistance gene, a trp promoter, a polyhistidine leader (including the first 6 STII codons, a polyhistidine sequence, and an enterokinase cleavage site), an NPOR coding region, a lambda transcription terminator, and an argU gene.

The ligation mixture is then used to transform the selected E. coli strain by the method described in Sambrook et al. (supra). Transformants are identified by their ability to grow on LB plates, and antibiotic-resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing.

The selected clones can be cultured overnight in a liquid medium (such as LB medium supplemented with antibiotics). The overnight culture can then be inoculated into a large-scale culture. The cells are then grown to the desired optical density, during which time the expression promoter is turned on.

After the cells are cultured for several hours, the cells can be harvested by centrifugation. The cell pellet harvested by centrifugation can be dissolved using various reagents known in the art, and the dissolved antibodies can then be purified using a metal chelate column under conditions that allow the antibodies to bind tightly.

Anti-PD-L1 antibodies can also be expressed in E. coli with a polyhistidine tag using the following procedure. First, DNA encoding the antibody is amplified using selected PCR primers. The primers contain restriction enzyme sites corresponding to those on the selected expression vector, as well as other useful sequences that provide efficient and reliable translation initiation, rapid purification on a metal chelate column, and proteolytic cleavage by enterokinase. The PCR-amplified, poly-His-tagged sequence was then ligated into an expression vector, which was used to transform an E. coli host based on strain 52 (W3110fuhA(tonA)lon galE rpoHts(htpRts)clpP(lacIq). First, the transformants were cultured in LB containing 50 mg/ml carbenicillin at 30°C with shaking until the OD600 reached 3-5. The culture was then grown in CRAP medium (prepared by mixing 3.57 g ( _NH4 ) _2SO4 , 0.71 g sodium citrate· _2H2O , 1.07 g KCl, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF, 110 mM MPOS pH 7.3, 0.55% (w/v) glucose, and 7 mM MgSO4 in 500 mL of water _{) .} The culture was diluted 50-100-fold in PBS (prepared from PBS) and cultured with shaking at 30°C for approximately 20-30 hours. Samples were removed for expression verification by SDS-PAGE analysis, and the bulk culture was centrifuged to pellet the cells. The cell pellet was frozen until purification and refolding.

The E. coli cell paste (6-10g sediment) from 0.5 to 1 liter of fermentation liquid is resuspended in 7M guanidine, 20mM Tris (pH 8) buffer of 10 times of volume (w/v). Add solid sodium sulfite and sodium tetrathionate to final concentrations of 0.1M and 0.02M, respectively, and the solution is stirred overnight at 4°C. This step denatures the protein, and all cysteine residues are blocked by sulfitolization. The solution is centrifuged 30 minutes at 40,000rpm in a Beckman ultracentrifuge. The supernatant is diluted with 3-5 times of volume of metal chelate column buffer (6M guanidine, 20mM Tris, pH 7.4) and filtered through a 0.22 micron filter membrane to clarify. Depending on the conditions, the clarified extract is loaded onto a 5ml Qiagen Ni-NTA metal chelate column balanced in metal chelate column buffer. The column was washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade) at pH 7.4. The protein was eluted with a buffer containing 250 mM imidazole. Fractions containing the desired protein were pooled and stored at 4°C. The protein concentration was estimated by its optical density at 280 nm using the extinction coefficient calculated based on its amino acid sequence.

The protein was refolded by slowly diluting the sample into freshly prepared refolding buffer consisting of 20 mM Tris (pH 8.6), 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine, and 1 mM EDTA. The refolding volume was selected so that the final protein concentration was between 50 and 100 μg/ml. The refolding solution was gently agitated at 4°C for 12-36 hours. The refolding reaction was quenched by adding TFA to a final concentration of 0.4% (pH approximately 3). Prior to further protein purification, the solution was filtered through a 0.22 μm filter and acetonitrile was added to a final concentration of 2-10%. The refolded protein was chromatographed on a Poros R1/H reverse phase column using 0.1% TFA as the running buffer and a gradient elution of 10 to 80% acetonitrile. Aliquots of fractions with A280 absorbance were analyzed on SDS polyacrylamide gels, and fractions containing homogeneous refolded protein were pooled. In general, the correctly refolded species of most proteins are eluted at the lowest acetonitrile concentrations because those species are the most compact and their hydrophobic interiors are protected from interacting with the reversed-phase resin. Aggregated species are often eluted at higher acetonitrile concentrations. In addition to resolving misfolded forms of proteins from the desired form, the reversed-phase step also removes endotoxins from the sample.

Fractions containing the desired, folded anti-PD-L1 antibody were pooled and the acetonitrile was removed with a gentle stream of nitrogen through the solution. The protein was formulated into 20 mM Hepes pH 6.8 containing 0.14 M sodium chloride and 4% mannose by dialysis or by gel filtration using G25 Superfine (Pharmacia) resin equilibrated in formulation buffer and sterile filtered.

Claims (61)

1. An isolated anti-PD-L1 antibody or its antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein: (a) The heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein: (i) HVR-H1 contains the amino acid sequence of SEQ ID NO:15; (ii) HVR-H2 contains the amino acid sequence of SEQ ID NO:16; (iii) HVR-H3 contains the amino acid sequence of SEQ ID NO:3; (b) The light chain variable region comprises HVR-L1, HVR-L2, and HVR-L3, wherein: (iv) HVR-L1 contains the amino acid sequence of SEQ ID NO:17; (v) HVR-L2 contains the amino acid sequence of SEQ ID NO:18; (vi) HVR-L3 contains the amino acid sequence of SEQ ID NO:19; (c) The amino acid sequence of the heavy chain variable region has at least 90% sequence identity with the amino acid sequence of the heavy chain variable region of SEQ ID NO:20; and (d) The amino acid sequence of the light chain variable region has at least 90% sequence identity with the amino acid sequence of the light chain variable region of SEQ ID NO:21. 2. The antibody or antigen-binding fragment of claim 1, wherein the amino acid sequence of the light chain variable region has at least 95% sequence identity with the amino acid sequence of the light chain variable region of SEQ ID NO: 21. 3. The antibody or antigen-binding fragment of claim 1, wherein the amino acid sequence of the light chain variable region has at least 99% sequence identity with the amino acid sequence of the light chain variable region of SEQ ID NO: 21. 4. The antibody or antigen-binding fragment of claim 1, wherein the amino acid sequence of the light chain variable region comprises the light chain variable region amino acid sequence of SEQ ID NO: 21. 5. The antibody or antigen-binding fragment of claim 1, wherein the amino acid sequence of the heavy chain variable region has at least 95% sequence identity with the amino acid sequence of the heavy chain variable region of SEQ ID NO: 20. 6. The antibody or antigen-binding fragment of claim 1, wherein the amino acid sequence of the heavy chain variable region has at least 99% sequence identity with the amino acid sequence of the heavy chain variable region of SEQ ID NO: 20. 7. The antibody or antigen-binding fragment of claim 4, wherein the amino acid sequence of the heavy chain variable region has at least 99% sequence identity with the amino acid sequence of the heavy chain variable region of SEQ ID NO: 20. 8. The antibody or antigen-binding fragment of any one of claims 1-7, further comprising a human constant region. 9. The antibody or antigen-binding fragment of claim 8, wherein the constant region is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4. 10. The antibody or antigen-binding fragment of claim 9, wherein the constant region is IgG1. 11. The antibody or antigen-binding fragment of any one of claims 1-7, having reduced or minimal effector function. 12. The antibody or antigen-binding fragment of claim 11, wherein the minimal effector function is generated by an Fc mutation of a worse effector. 13. The antibody or antigen-binding fragment of claim 12, wherein the Fc mutation of the worse effector is N297A. 14. The antibody or antigen-binding fragment of claim 12, wherein the Fc mutation of the worse effector is D265A and N297A. 15. The antibody or antigen-binding fragment of claim 11, wherein the minimal effector function is generated by deglycosylation. 16. A composition comprising an anti-PD-L1 antibody or antigen-binding fragment of any one of claims 1-15 and at least one pharmaceutically acceptable carrier. 17. An isolated nucleic acid encoding an antibody of any one of claims 1-15. 18. A vector comprising the nucleic acid of claim 17. 19. A host cell comprising the vector of claim 18. 20. The host cell of claim 19, wherein it is eukaryotic. 21. The host cell of claim 20, which is mammalian. 22. The host cell of claim 21 is a Chinese hamster ovary (CHO) cell. 23. A method for preparing an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising culturing a host cell of any one of claims 19-22 under conditions suitable for expressing a vector encoding an anti-PD-L1 antibody or an antigen-binding fragment, and recovering the antibody or antigen-binding fragment. 24. An article comprising an antibody or antigen-binding fragment of any one of claims 1-15. 25. An article comprising the composition of claim 16. 26. The article of claim 24 or 25, further comprising at least one chemotherapeutic agent. 27. The article of claim 26, wherein the chemotherapeutic agent is gemcitabine. 28. The article of claim 26, wherein the chemotherapeutic agent is an anti-VEGF antibody. 29. The article of claim 28, wherein the anti-VEGF antibody is bevacizumab. 30. The article of claim 24 or 25, further comprising at least one agonist of a positive co-stimulatory molecule. 31. The article of claim 24 or 25, further comprising a B7 negative co-stimulatory antagonist. 32. The article of claim 24 or 25, further comprising at least one B7-family co-stimulatory molecule. 33. The article of claim 24 or 25, further comprising at least one vaccine. 34. Use of an effective amount of the antibody or antigen-binding fragment of any one of claims 1-15 in the preparation of a medicament for treating cancer. 35. Use of an effective amount of the composition of claim 16 in the preparation of a medicament for treating cancer. 36. The application of claim 34 or 35, wherein the cancer is selected from the group consisting of: breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma, bladder cancer, kidney cancer, liver cancer, salivary gland cancer, stomach cancer, glioma, thyroid cancer, thymic cancer, epithelial cancer, head and neck cancer, gastric cancer, and pancreatic cancer. 37. The application of claim 34 or 35, wherein the drug is used in combination with a chemotherapeutic agent. 38. The application of claim 37, wherein the cancer is selected from the group consisting of: breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma, bladder cancer, kidney cancer, liver cancer, salivary gland cancer, stomach cancer, glioma, thyroid cancer, thymic cancer, epithelial cancer, head and neck cancer, stomach cancer, and pancreatic cancer. 39. The application of claim 37, wherein the chemotherapeutic agent is an anti-VEGF antibody. 40. The application of claim 39, wherein the anti-VEGF antibody is bevacizumab. 41. The application of claim 37, wherein the chemotherapeutic agent is FOLFOX. 42. The application of claim 41, wherein FOLFOX is a combination of oxaliplatin, 5-FU and leucovovin. 43. The application of claim 37, wherein the chemotherapeutic agent is oxaliplatin. 44. The application of claim 37, wherein the chemotherapeutic agent is a RAF inhibitor. 45. Use of an effective amount of the antibody or antigen-binding fragment of any one of claims 1-15 in the preparation of a medicament for treating infections. 46. Use of an effective amount of the composition of claim 16 in the preparation of a medicament for treating infections. 47. The application of claim 45 or 46, wherein the drug is used in combination with an antibiotic. 48. The application of claim 47, wherein the antibiotic is an antiviral agent. 49. The application of claim 48, wherein the antiviral agent is a reverse transcriptase inhibitor. 50. The application of claim 49, wherein the reverse transcriptase inhibitor is a polymerase inhibitor. 51. The application of claim 48, wherein the antiviral agent is a protease inhibitor. 52. The application of claim 45 or 46, wherein the drug is used in combination with at least one vaccine. 53. The application of claim 45 or 46, wherein the infection is chronic. 54. The application of claim 53, wherein the chronic infection is persistent. 55. The application of claim 53, wherein the chronic infection is latent. 56. The application of claim 53, wherein the chronic infection is slow. 57. The application of claim 45 or 46, wherein the infection is caused by a pathogen selected from the group consisting of bacteria, viruses, fungi, and protozoa. 58. The application of claim 57, wherein the pathogen is a bacterium and the drug is used in combination with an antimicrobial agent. 59. The application of claim 57, wherein the pathogen is a virus and the drug is used in combination with an antiviral agent. 60. The application of claim 57, wherein the pathogen is a fungus and the drug is used in combination with an antifungal agent. 61. The application of claim 57, wherein the pathogen is a protozoan and the drug is used in combination with an antiprotozoan agent.