Bispecific PD-L1 and CD40 binding molecules and uses thereof

Abstract

Provided herein are bispecific antigen-binding molecules that bind to PD-L1 and / or CD40. Further provided herein are therapeutic uses and methods of producing such molecules, as well as related pharmaceutical compositions, nucleic acid molecules, expression vectors, and host cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Application No. 63 / 478,799, filed January 6, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Reference The contents of the electronic sequence listing (APVO_070_01WO_SeqList_ST26.xml, size: 536,960 bytes, and creation date: January 3, 2024) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to bispecific antigen-binding molecules and therapeutic uses of such molecules. [Background technology]

[0004] Despite major advances in the development of cancer therapies, cancer remains a global health burden. Approximately 2 million new cancer cases and 609,820 cancer-related deaths are predicted in the United States in 2023 (Siegal et al., CA Cancer J Clin. 73:17-48 (2023)). Therefore, new, effective cancer therapies, particularly those that are less toxic and more targeted than traditional chemotherapy and radiation, are needed. Novel therapies that harness a patient's own immune system are attractive because tumor-specific T cells have been shown to both eliminate tumors and mount memory responses to prevent recurrence.

[0005] Immune cells have a series of proteins on their surface that are intended to enhance (co-stimulatory) or suppress (co-inhibitory) the immune response when triggered. A strengthened immune response is necessary to adequately combat foreign pathogens through cell proliferation, differentiation, and the generation of memory cells. During cell activation, the interaction of costimulatory molecules between immune cell types plays a major role in the nature of the immune response generated.

[0006] Suppressing the immune response is beneficial in preventing overactivation of the immune system, which can lead to adverse effects such as allergies and asthma. These proteins are therefore called brakes or checkpoints. Checkpoint proteins are a way for the immune system to communicate between many immune and non-immune cells. Cancer cells hijack this pathway to avoid detection and killing by immune cells.

[0007] Currently, checkpoint inhibitors targeting several inhibitory pathways are under development. The most successful targets target the PD-1 / PD-L1 pathway. PD-L1 is expressed on T cells, B cells, DCs, and macrophages at rest and is upregulated upon activation. Its normal function is to regulate the balance between T cell activation and tolerance through interactions with its receptors, PD-1 and CD80. Furthermore, PD-L1 is expressed in a wide range of cancers and is associated with reduced patient survival and poor prognosis. Antibodies that block the interaction between PD-1 and PD-L1 can ablate immunosuppressive disruption and enhance tumor cytotoxicity.

[0008] Stimulatory receptors are utilized to enhance immune responses and generate mature cells. CD40 is one such molecule expressed on antigen-presenting cells (B cells, macrophages, and DCs). Interaction with its ligand (CD40L) on activated CD4 T cells activates APCs to generate mature cells, which then induce antigen, upregulation of costimulatory molecules, and secretion of inflammatory cytokines, leading to the priming of CD8 T cells. Summary of the Invention

[0009] Provided herein is a PD-L1-binding polypeptide that specifically binds to human PD-L1, the PD-L1-binding polypeptide comprising, in amino-terminal to carboxyl-terminal or carboxyl-terminal to amino-terminal order: (a) a first binding domain, (b) a hinge region, (c) an immunoglobulin constant region, and (d) a second binding domain, wherein the first binding domain is a PD-L1-binding domain and the second binding domain binds to an immunostimulatory protein, or the first binding domain binds to an immunostimulatory protein and the second binding domain is a PD-L1-binding domain. In some embodiments, the immunostimulatory protein is CD40, 4-1BB, CD3, or OX40.

[0010] In some embodiments of the PD-L1-binding domain, the PD-L1-binding domain comprises (i) an immunoglobulin heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3, and (ii) an immunoglobulin light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3. In some embodiments of the PD-L1-binding domain, HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 5, LCDR2 comprises SEQ ID NO: 6, and LCDR3 comprises SEQ ID NO: 7. In some embodiments, the VH comprises SEQ ID NO: 4 and the VL comprises SEQ ID NO: 8. In some embodiments, the PD-L1-binding domain comprises SEQ ID NO: 9.

[0011] Further provided herein is a CD40-binding polypeptide that specifically binds to human CD40, comprising, in amino-terminal to carboxyl-terminal or carboxyl-terminal to amino-terminal order: (a) a first binding domain, (b) a hinge region, (c) an immunoglobulin constant region, and (d) a second binding domain, wherein the first binding domain is a CD40-binding domain and the second binding domain binds to a tumor-associated antigen, or the first binding domain binds to a tumor-associated antigen and the second binding domain is a CD40-binding domain. In some embodiments, the tumor-associated antigen is PD-L1, ROR1, or EGFR.

[0012] In some embodiments, the CD40 binding domain comprises (i) an immunoglobulin heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3, and (ii) an immunoglobulin light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3.

[0013] In some embodiments of the CD40 binding domain, (a) HCDR1 comprises SEQ ID NO: 10, HCDR2 comprises SEQ ID NO: 11, HCDR3 comprises SEQ ID NO: 12, LCDR1 comprises SEQ ID NO: 14, LCDR2 comprises SEQ ID NO: 15, and LCDR3 comprises SEQ ID NO: 16; (b) HCDR1 comprises SEQ ID NO: 10, HCDR2 comprises SEQ ID NO: 23, HCDR3 comprises SEQ ID NO: 12, LCDR1 comprises SEQ ID NO: 14, LCDR2 comprises SEQ ID NO: 15, and LCDR3 comprises SEQ ID NO: 16; or (c) HCDR1 , HCDR1 comprises SEQ ID NO: 10, HCDR2 comprises SEQ ID NO: 11, HCDR3 comprises SEQ ID NO: 31, LCDR1 comprises SEQ ID NO: 14, LCDR2 comprises SEQ ID NO: 15, and LCDR3 comprises SEQ ID NO: 16; (d) HCDR1 comprises SEQ ID NO: 10, HCDR2 comprises SEQ ID NO: 23, HCDR3 comprises SEQ ID NO: 31, LCDR1 comprises SEQ ID NO: 14, LCDR2 comprises SEQ ID NO: 15, and LCDR3 comprises SEQ ID NO: 16; or (e) HCDR1 comprises SEQ ID NO: 10, HCDR2 comprises SEQ ID NO: 11, HCDR3 comprises SEQ ID NO: 12, LCDR1 comprises SEQ ID NO: 37, LCDR2 comprises SEQ ID NO: 15, and LCDR3 comprises SEQ ID NO: 16; (f) HCDR1 comprises SEQ ID NO: 10, HCDR2 comprises SEQ ID NO: 11, and HCDR3 comprises SEQ ID NO: 12, LCDR1 comprises SEQ ID NO: 41, LCDR2 comprises SEQ ID NO: 15, and LCDR3 comprises SEQ ID NO: 16; or (g) HCDR1 comprises SEQ ID NO: 10, HCDR2 comprises SEQ ID NO: 45, HCDR3 comprises SEQ ID NO: 12, LCDR1 comprises SEQ ID NO: 14, and LCDR 2 comprises SEQ ID NO: 15 and LCDR3 comprises SEQ ID NO: 16; (h) HCDR1 comprises SEQ ID NO: 10, HCDR2 comprises SEQ ID NO: 49, HCDR3 comprises SEQ ID NO: 12, LCDR1 comprises SEQ ID NO: 14, LCDR2 comprises SEQ ID NO: 15 and LCDR3 comprises SEQ ID NO: 16; or (i) HCDR1 comprises SEQ ID NO: 10, HCDR2 comprises SEQ ID NO: 23, HCDR3 comprises SEQ ID NO: 31, LCDR1 comprises SEQ ID NO: 37, LCDR2 comprises SEQ ID NO: 15 and LCDR3 comprises SEQ ID NO: 16.

[0014] In some embodiments of the CD40 binding domain, (a) the VH comprises SEQ ID NO: 13 and the VL comprises SEQ ID NO: 17; (b) the VH comprises SEQ ID NO: 24 and the VL comprises SEQ ID NO: 17; (c) the VH comprises SEQ ID NO: 27 and the VL comprises SEQ ID NO: 28; (d) the VH comprises SEQ ID NO: 32 and the VL comprises SEQ ID NO: 17; (e) the VH comprises SEQ ID NO: 34 and the VL comprises SEQ ID NO: 17; or (f) the VH comprises (g) the VH comprises SEQ ID NO: 13 and the VL comprises SEQ ID NO: 38; (h) the VH comprises SEQ ID NO: 46 and the VL comprises SEQ ID NO: 17; (i) the VH comprises SEQ ID NO: 50 and the VL comprises SEQ ID NO: 17; (j) the VH comprises SEQ ID NO: 484 and the VL comprises SEQ ID NO: 485; or (k) the VH comprises SEQ ID NO: 484 and the VL comprises SEQ ID NO: 28.

[0015] In some embodiments, the CD40 binding domain comprises SEQ ID NO: 18, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 486, or SEQ ID NO: 488.

[0016] Provided herein are binding polypeptides that specifically bind to human PD-L1 and human CD40, the binding polypeptides comprising, in amino-terminal to carboxyl-terminal or carboxyl-terminal to amino-terminal order: (a) a first binding domain, (b) a hinge region, (c) an immunoglobulin constant region, and (d) a second binding domain, wherein the first binding domain is a PD-L1-binding domain and the second binding domain is a CD40-binding domain, or the first binding domain is a CD40-binding domain and the second binding domain is a PD-L1-binding domain.

[0017] Provided herein is a binding polypeptide, wherein the PD-L1-binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; and the CD40-binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 11, and an HCDR3 comprising SEQ ID NO: 12, and a VL comprising an LCDR1 comprising SEQ ID NO: 14, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO: 16.

[0018] Provided herein is a binding polypeptide, wherein the PD-L1-binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; and the CD40-binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 23, and an HCDR3 comprising SEQ ID NO: 12, and a VL comprising an LCDR1 comprising SEQ ID NO: 14, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO: 16.

[0019] Provided herein is a binding polypeptide, wherein the PD-L1-binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; and the CD40-binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 11, and an HCDR3 comprising SEQ ID NO: 31, and a VL comprising an LCDR1 comprising SEQ ID NO: 14, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO: 16.

[0020] Provided herein is a binding polypeptide, wherein the PD-L1-binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 7, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; and the CD40-binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 23, and an HCDR3 comprising SEQ ID NO: 31, and a VL comprising an LCDR1 comprising SEQ ID NO: 14, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO: 16.

[0021] Provided herein is a binding polypeptide, wherein the PD-L1-binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; and the CD40-binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 11, and an HCDR3 comprising SEQ ID NO: 12, and a VL comprising an LCDR1 comprising SEQ ID NO: 37, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO: 16.

[0022] Provided herein is a binding polypeptide, wherein the PD-L1-binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; and the CD40-binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 11, and an HCDR3 comprising SEQ ID NO: 12, and a VL comprising an LCDR1 comprising SEQ ID NO: 41, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO: 16.

[0023] Provided herein is a binding polypeptide, wherein the PD-L1-binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; and the CD40-binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 45, and an HCDR3 comprising SEQ ID NO: 12, and a VL comprising an LCDR1 comprising SEQ ID NO: 14, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO: 16.

[0024] Provided herein is a binding polypeptide, wherein the PD-L1-binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; and the CD40-binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 49, and an HCDR3 comprising SEQ ID NO: 12, and a VL comprising an LCDR1 comprising SEQ ID NO: 14, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO: 16.

[0025] Provided herein is a binding polypeptide, wherein the PD-L1-binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; and the CD40-binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 23, and an HCDR3 comprising SEQ ID NO: 31, and a VL comprising an LCDR1 comprising SEQ ID NO: 37, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO: 16.

[0026] Provided herein is a binding polypeptide, wherein (a) the PD-L1-binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40-binding domain comprises a VH comprising SEQ ID NO: 13 and a VL comprising SEQ ID NO: 17; (b) the PD-L1-binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40-binding domain comprises a VH comprising SEQ ID NO: 24 and a VL comprising SEQ ID NO: 17; or (c) the PD-L1-binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40-binding domain comprises a VH comprising SEQ ID NO: 24 and a VL comprising SEQ ID NO: 17. (d) the PD-L1 binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40 binding domain comprises a VH comprising SEQ ID NO: 32 and a VL comprising SEQ ID NO: 17; (e) the PD-L1 binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40 binding domain comprises a VH comprising SEQ ID NO: 34 and a VL comprising SEQ ID NO: 17; or (f) the PD-L1 binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8. (g) the PD-L1 binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40 binding domain comprises a VH comprising SEQ ID NO: 13 and a VL comprising SEQ ID NO: 38; (g) the PD-L1 binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40 binding domain comprises a VH comprising SEQ ID NO: 13 and a VL comprising SEQ ID NO: 42; (h) the PD-L1 binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40 binding domain comprises a VH comprising SEQ ID NO: 46 and a VL comprising SEQ ID NO: 17; or (i) the PD-L1 binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: (j) the PD-L1 binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40 binding domain comprises a VH comprising SEQ ID NO: 50 and a VL comprising SEQ ID NO: 17; or (k) the PD-L1 binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40 binding domain comprises a VH comprising SEQ ID NO: 484 and a VL comprising SEQ ID NO: 28.

[0027] Provided herein is a binding polypeptide, wherein (a) the PD-L1-binding domain comprises SEQ ID NO: 9 and the CD40-binding domain comprises SEQ ID NO: 18; (b) the PD-L1-binding domain comprises SEQ ID NO: 9 and the CD40-binding domain comprises SEQ ID NO: 25; (c) the PD-L1-binding domain comprises SEQ ID NO: 9 and the CD40-binding domain comprises SEQ ID NO: 29; (d) the PD-L1-binding domain comprises SEQ ID NO: 9 and the CD40-binding domain comprises SEQ ID NO: 33; (e) the PD-L1-binding domain comprises SEQ ID NO: 9 and the CD40-binding domain comprises SEQ ID NO: 35; or (f) the PD-L1-binding domain comprises SEQ ID NO: 9. (i) the PD-L1 binding domain comprises SEQ ID NO: 9 and the CD40 binding domain comprises SEQ ID NO: 51; (j) the PD-L1 binding domain comprises SEQ ID NO: 9 and the CD40 binding domain comprises SEQ ID NO: 486; or (k) the PD-L1 binding domain comprises SEQ ID NO: 9 and the CD40 binding domain comprises SEQ ID NO: 488.

[0028] Provided herein are binding polypeptides comprising SEQ ID NO:19, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:280, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:487, or SEQ ID NO:489.

[0029] In some embodiments, the hinge is an immunoglobulin hinge.

[0030] In some embodiments, the immunoglobulin constant region comprises immunoglobulin CH2 and CH3 domains of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD.

[0031] In some embodiments, the immunoglobulin constant region comprises a human IgG1 CH2 domain comprising an amino acid substitution at one or more of the following residues according to the EU numbering system: E233, L234, L235, G236, G237, E318, K320, K322. In some embodiments, the amino acid substitution at residue E233 is E233P. In some embodiments, the amino acid substitution at residue L234 is selected from the group consisting of L234A and L234V. In some embodiments, the amino acid substitution at residue L235 is L235A. In some embodiments, the amino acid substitution at residue G237 is G237A. In some embodiments, the amino acid substitution at E318 is E318A. In some embodiments, the amino acid substitution at K320 is K320A. In some embodiments, the amino acid substitution at K322 is K322A. In some embodiments, residue G236 (according to the EU numbering system) is deleted.

[0032] Provided herein are dimeric proteins comprising the binding polypeptides disclosed herein. In some embodiments, the dimeric protein is a homodimer.

[0033] Provided herein are compositions comprising a binding polypeptide disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient. Provided herein are compositions comprising a dimeric protein disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0034] Provided herein is a nucleic acid molecule comprising a nucleotide sequence encoding a binding polypeptide disclosed herein. In some embodiments, the nucleotide sequence comprises SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:259, or SEQ ID NO:260. Provided herein is an expression vector comprising a nucleic acid molecule disclosed herein. Provided herein is a recombinant host cell comprising a nucleic acid molecule disclosed herein or an expression vector disclosed herein.

[0035] Provided herein are methods for producing a binding polypeptide, comprising culturing a recombinant host cell disclosed herein under conditions in which a nucleic acid molecule is expressed, thereby producing a binding polypeptide, and isolating the binding polypeptide from the host cell or culture.

[0036] Provided herein are methods for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a binding polypeptide, dimeric protein, or composition disclosed herein.

[0037] Provided herein are methods for treating a symptom of cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a binding polypeptide, dimeric protein, or composition disclosed herein.

[0038] In any of the embodiments of the methods of treatment provided herein, the cancer may be a solid tumor. In any of the embodiments of the methods of treatment provided herein, the cancer may express PD-L1.

[0039] In any of the embodiments of the treatment methods provided herein, the cancer may be head and neck cancer, melanoma, lung cancer, brain cancer, thymus cancer, breast cancer, liver cancer, pancreatic cancer, kidney cancer, ovarian cancer, bladder cancer, gastrointestinal cancer, or colon cancer.

[0040] Provided herein is a binding polypeptide, dimeric protein, or composition disclosed herein for use as a medicament. [Brief explanation of the drawings]

[0041] [Figure 1]Figure 1 illustrates an amino acid alignment showing the humanization progress from murine anti-PD-L1 clone 5F9 (PDL01034) to the fully human antibody sequence of the PDL01152 molecule. Alignment of the VH and VL chains to the closest human immunoglobulin germline V and J segments is shown. CDRs (Kabat definition) are shown in bold, underlined font. In the VH alignment, the sequences, from top to bottom, are SEQ ID NOs: 288-292 and IGHV3-48+IGHJ4 (SEQ ID NO: 278). In the VL alignment, the sequences, from top to bottom, are SEQ ID NOs: 293-297 and IGKV1-39+IGKJ2 (SEQ ID NO: 279). [Figure 2] Figures A-F show the binding of scFv anti-PD-L1 constructs to human (A and D), cynomolgus monkey (B and E), and parental PD-L1-expressing CHOK1SV (C and F) cells. Serial dilutions of N-terminal anti-PD-L1 constructs were incubated with transfected target cells, followed by labeling with a SULFO-TAG-conjugated goat anti-human IgG secondary antibody. Binding was quantified by MSD (Meso Scale Discovery instrument). The y-axis displays the signal in electrochemiluminescence units relative to the background signal. (See Example 7.) [Figure 3] A-B show the binding of anti-PD-L1 lead PDL01034 (A) or PDL01036 (B) to various cell lines that express PD-L1 or were negative controls to assess non-specific binding. Serial dilutions of N-terminal anti-PD-L1 constructs were incubated with transfected target cells or tumor cell lines, followed by labeling with a SULFO-TAG-conjugated goat anti-human IgG secondary antibody. Binding was quantified by MSD (Meso Scale Discovery instrument). The y-axis displays the signal in electrochemiluminescence units relative to the background signal. (See Example 8.) [Figure 4]Figures A-B show the ability of hybridoma antibody clones 1D5, 2H7, and 5B9 to block CD40 receptor-CD40 ligand binding on cells. Two sets of experiments were performed. In the first assay (A), CD40-expressing cells were saturated with His-tagged CD40L (BPS Bioscience) followed by ADAPTIR binding. Cells were incubated with PE-labeled goat anti-hu IgG F(ab')2 (Jackson). In the second assay (B), CD40-expressing cells were saturated with antibody followed by CD40L and detected using PE-labeled anti-His (Biolegend). All cells were washed and analyzed by flow cytometry. (See Example 12.) [Figure 5] Panels A-B show the binding of anti-PD-L1 and anti-CD40 bispecific proteins with various structures and valencies. Serial dilutions of the constructs were incubated with CHOK1SV cells transfected with CD40 (A) or PD-L1 (B) and then labeled with a fluorescently conjugated goat-α-human Fc secondary antibody. The y-axis displays mean fluorescence intensity units (MFI). (See Example 13.) [Figure 6] Figure 1 shows the PD-L1 / PD-1 blocking ability of anti-PD-L1 and anti-CD40 bispecific proteins with various structures and valencies. Serial dilutions of the constructs were incubated with a human PD-1 NFAT luciferase reporter Jurkat line (BPS) and TCR activator PD-L1-expressing CHO target cells (BPS). After 6 hours, Bio-Glo luciferase reagent (Promega) was added, and luminescence was read using a MicroBeta 2450 Microplate Counter (Perkin Elmer). The y-axis displays light units relative to the background signal. (See Example 13.) [Figure 7]Panels A-B show the CD40 signaling ability of anti-PD-L1 and anti-CD40 bispecific proteins with various structures and valencies. Serial dilutions of the constructs were incubated with human CD40 / NFκB HEK293 luciferase reporter cells (BPS) and PD-L1-expressing (A) or parental (B) CHOK1SV target cells. After 6 hours, Bio-Glo luciferase reagent (Promega) was added and luminescence was read using a MicroBeta 2450 Microplate Counter (Perkin Elmer). The y-axis displays light units relative to the background signal. (See Example 13.) [Figure 8A] Figure 1 illustrates an amino acid alignment showing the progress of humanization from murine anti-CD40 clone 1D5 (CD401016) to the fully humanized antibody sequence CD401164. Alignment of the VH chain to the closest human immunoglobulin germline V and J segments is shown. CDRs (Kabat definition) are shown in bold, underlined font. The sequences, from top to bottom, are SEQ ID NOS: 298-318 and 281. [Figure 8B] Illustrates an amino acid alignment showing the progress of humanization from murine anti-CD40 clone 1D5 (CD401016) to the fully humanized antibody sequence CD401164. Alignment of the VL chain to the closest human immunoglobulin germline V and J segments is shown. CDRs (Kabat definition) are shown in bold, underlined font. The sequences, from top to bottom, are SEQ ID NOS: 319-339 and 282. [Figure 8C] Figure 1 illustrates an amino acid alignment showing the progress of humanization from murine anti-CD40 clone 1D5 (CD401016) to the fully humanized antibody sequence CD401242. Alignment of the VH chain to the closest human immunoglobulin germline V and J segments is shown. CDRs (Kabat definition) are shown in bold underlined text. The sequences, from top to bottom, are SEQ ID NOS: 340-378 and 283. [Figure 8D]Figure 1 illustrates an amino acid alignment showing the progress of humanization from murine anti-CD40 clone 1D5 (CD401016) to the fully humanized antibody sequence CD401242. Alignment of the VL chain to the closest human immunoglobulin germline V and J segments is shown. CDRs (Kabat definition) are shown in bold, underlined font. The sequences, from top to bottom, are SEQ ID NOS: 379-417 and 284. [Figure 9A] Figure 1 illustrates an amino acid alignment showing the progress of humanization from the mouse anti-CD40 clone 5B9 (CD401018) clone to the fully humanized antibody sequence CD401133. Alignment of the VH chain to the closest human immunoglobulin germline V and J segments is shown. CDRs (Kabat definition) are shown in bold, underlined font. The sequences, from top to bottom, are SEQ ID NOS: 418-435 and 285. [Figure 9B] Figure 1 illustrates an amino acid alignment showing the progression of humanization from the mouse anti-CD40 clone 5B9 (CD401018) clone to the fully humanized antibody sequence CD401133. Figure 2 illustrates an amino acid alignment showing the progression of humanization from the mouse clone to the fully humanized antibody sequence CD401133. Alignment of the VL chain to the closest human immunoglobulin germline V and J segments is shown. CDRs (Kabat definition) are shown in bold, underlined font. The sequences, from top to bottom, are SEQ ID NOs: 436-453 and 286. [Figure 10A] Figure 1 shows multiple binding of humanized anti-CD40 scFv antibodies. Serial dilutions of N-terminal scFv constructs were incubated with CHOK1SV cells transfected with human CD40 and then labeled with a fluorescently conjugated goat-α-human Fc secondary antibody. The y-axis displays mean fluorescence intensity units (MFI). (See Example 21.) [Figure 10B] Figure 1 shows multiple binding of humanized anti-CD40 scFv antibodies. Serial dilutions of N-terminal scFv constructs were incubated with CHOK1SV cells transfected with human CD40 and then labeled with a fluorescently conjugated goat-α-human Fc secondary antibody. The y-axis displays mean fluorescence intensity units (MFI). (See Example 21.) [Figure 10C]Figure 1 shows multiple binding of humanized anti-CD40 scFv antibodies. Serial dilutions of N-terminal scFv constructs were incubated with CHOK1SV cells transfected with human CD40 and then labeled with a fluorescently conjugated goat-α-human Fc secondary antibody. The y-axis displays mean fluorescence intensity units (MFI). (See Example 21.) [Figure 10D] Figure 1 shows multiple binding of humanized anti-CD40 scFv antibodies. Serial dilutions of N-terminal scFv constructs were incubated with CHOK1SV cells transfected with human CD40 and then labeled with a fluorescently conjugated goat-α-human Fc secondary antibody. The y-axis displays mean fluorescence intensity units (MFI). (See Example 21.) [Figure 10E] Figure 1 shows multiple binding of humanized anti-CD40 scFv antibodies. Serial dilutions of N-terminal scFv constructs were incubated with CHOK1SV cells transfected with human CD40 and then labeled with a fluorescently conjugated goat-α-human Fc secondary antibody. The y-axis displays mean fluorescence intensity units (MFI). (See Example 21.) [Figure 11] A-B show the binding of anti-PD-L1 and anti-CD40 ADAPTIR antibodies to tumor cell lines. Serial dilutions of ADAPTIR constructs were incubated with Daudi or MDA-MB-231 tumor cells, followed by labeling with a fluorescently conjugated goat-α-human Fc secondary antibody. The y-axis displays mean fluorescence intensity units (MFI). (See Example 22.) [Figure 12A] Figure 2 shows the binding of optimized anti-PD-L1 and anti-CD40 ADAPTIR antibodies to multiple cell lines. Serial dilutions of ADAPTIR constructs were incubated with human CHOK1SV / CD40 cells. After binding, the cells were then labeled with a fluorescently conjugated goat-α-human Fc secondary antibody. The y-axis displays mean fluorescence intensity units (MFI). (See Example 24.) [Figure 12B]Figure 2 shows the binding of optimized anti-PD-L1 and anti-CD40 ADAPTIR antibodies to multiple cell lines. Serial dilutions of ADAPTIR constructs were incubated with cynomolgus monkey CHOK1SV / CD40 cells. After binding, the cells were then labeled with a fluorescently conjugated goat-α-human Fc secondary antibody. The y-axis displays mean fluorescence intensity units (MFI). (See Example 24.) [Figure 12C] Figure 2 shows the binding of optimized anti-PD-L1 and anti-CD40 ADAPTIR antibodies to multiple cell lines. Serial dilutions of ADAPTIR constructs were incubated with Daudi cells. After binding, the cells were then labeled with a fluorescently conjugated goat-α-human Fc secondary antibody. The y-axis displays mean fluorescence intensity units (MFI). (See Example 24.) [Figure 12D] Figure 2 shows the binding of optimized anti-PD-L1 and anti-CD40 ADAPTIR antibodies to multiple cell lines. Serial dilutions of ADAPTIR constructs were incubated with human CHOK1SV / PD-L1 cells. After binding, the cells were then labeled with a fluorescently conjugated goat-α-human Fc secondary antibody. The y-axis displays mean fluorescence intensity units (MFI). (See Example 24.) [Figure 12E] Figure 2 shows the binding of optimized anti-PD-L1 and anti-CD40 ADAPTIR antibodies to multiple cell lines. Serial dilutions of ADAPTIR constructs were incubated with parental CHOK1SV cells. After binding, the cells were then labeled with a fluorescently conjugated goat-α-human Fc secondary antibody. The y-axis displays mean fluorescence intensity units (MFI). (See Example 24.) [Figure 13]Panels A-C show the functionality of anti-PD-L1 and anti-CD40 ADAPTIR antibodies. (A) In the PD-L1 / PD-1 blocking assay, serial dilutions of ADAPTIR were combined and incubated with human PD-1 NFκB luciferase reporter Jurkat cells (BPS) and TCR activator PD-L1-expressing CHO target cells (BPS). (B and C) In the CD40 reporter assay, serial dilutions of ADAPTIR were combined with human CD40 / NFκB HEK293 luciferase reporter cells (BPS) and PD-L1-expressing (B) or parental CHOK1SV (C) target cells. After 6 hours, Bio-Glo luciferase reagent (Promega) was added, and luminescence was read using a MicroBeta 2450 Microplate Counter (Perkin Elmer). The y-axis represents light units relative to the background signal. (See Example 25.) [Figure 14] Panels A-B show the activity of the optimized anti-PD-L1 x anti-CD40 ADAPTIR bispecific in targeting and killing tumors in vitro. SCC152 (ATCC) cells were sequentially transduced with lentivirus to express Nuclight Orange (NLO, Sartorius), human PD-L1, and EBV proteins (Vectorbuilder). Panel A shows target cells antibiotically selected to be triple-positive for NLO, human PD-L1, and EBV proteins using flow cytometry on a BD FACSymphony. For the cytotoxicity assay, M2 macrophages, immature DCs, and EBV-specific T cells were prepared separately prior to the assay. Adherent target SCC152 cells expressing NLO, PD-L1, and EBV proteins were plated 1 day before the assay setup. Titered ADAPTIR or control macrophages, DCs, and T cells were then added to the SCC152 target cells on assay day 0. Assay plates were imaged every 8 hours on an Incucyte reader (Sartorius) over the course of 6 days. B, Graphical representation of the number of viable SCC152 tumor cells upon addition of 0.25 nM of test or control molecule (see Example 26). [Figure 15] The protein sequence alignment of the CD40 extracellular domain used for epitope mapping of the 1D5 antibody is shown. All proteins were constructed with an N-terminal 2xFLAG tag and the human CD40 transmembrane and intracellular domains and expressed in CHO cells. Binding was examined by flow cytometry using anti-FLAG and 1D5 antibodies. Binding signals for all constructs were normalized to the FLAG and human CD40 signals and are shown on the right as % binding to human CD40. The sequences, from top to bottom, are SEQ ID NOs: 287 and 454-483. [Figure 16] Panels A-B show the functionality of anti-CD40 ADAPTIR bispecifics targeting either the ROR1 or EGFR tumor-associated antigens. For the CD40 reporter assay, serially diluted ADAPTIR was combined with human CD40 / NFκB HEK293 luciferase reporter cells (BPS) and tumor target cells. In panel A, the bispecific was generated using the anti-ROR1 binding domain. The targeted cell line used in the assay was Kasumi-2. In panel B, the bispecific was generated using the anti-EGFR binding domain. The targeted cell line used in the assay was PC-3. After 6 hours of incubation, Bio-Glo luciferase reagent (Promega) was added, and luminescence was read using a MicroBeta 2450 Microplate Counter (Perkin Elmer). The y-axis displays light units relative to the background signal. (See Example 30.) [Figure 17]Panels A-C show the functionality of anti-PD-L1 ADAPTIR bispecifics in inducing NFκB signaling of CD3, OX40, or 4-1BB. (A) Jurkat cells harboring a luciferase reporter gene under the control of the NFκB promoter were cocultured with the CD3-binding bispecific construct PC401020 to induce target-dependent activation of CD3. (B) In the OX40 reporter assay, Jurkat cells transfected to express human OX40 and a reporter gene were cocultured with the OX40-binding bispecific construct PC401022 to induce target-dependent activation of OX40. (C) In the 4-1BB reporter assay, Jurkat cells transfected to express human 4-1BB and a reporter gene were cocultured with the 4-1BB-binding bispecific construct PC401021 to induce target-dependent activation of 4-1BB. Serially diluted constructs were incubated with target cells and NFκB reporter cell lines for 5 hours, followed by addition of Bio-Glo. The y-axis displays RLU (see Example 31). [Figure 18] Figures A-D show the binding of optimized anti-PD-L1 and anti-CD40 ADAPTIR antibodies with stabilized binding domains to multiple cell lines. Serial dilutions of ADAPTIR constructs were incubated with Daudi tumor (A), cynomolgus monkey CHOK1SV / CD40 (B), human CHOK1SV / PD-L1 (C), or parental (D) CHOK1SV cells. After binding, cells were then labeled with a fluorescently conjugated goat-α-human Fc secondary antibody. The y-axis displays mean fluorescence intensity units (MFI). (See Example 32.) [Figure 19]Panels A-B show the functionality of anti-PD-L1 and anti-CD40 ADAPTIR antibodies with stabilized binding domains. For the CD40 reporter assay, serial dilutions of ADAPTIR were combined with human CD40 / NFκB HEK293 luciferase reporter cells (BPS) and PD-L1-expressing CHOK1SV target cells (A). For the PD-L1 / PD-1 blocking assay, serial dilutions of ADAPTIR were combined and incubated with human PD-1 NFAT luciferase reporter Jurkat cells (BPS) and TCR activator PD-L1-expressing CHO target cells (BPS) (B). After 6 hours, Bio-Glo luciferase reagent (Promega) was added, and luminescence was read using a MicroBeta 2450 Microplate Counter (Perkin Elmer). The y-axis represents light units relative to the background signal. (See Example 33.) [Figure 20] Panels A-B show the functionality and cross-linking requirements of anti-PD-L1 and anti-CD40 ADAPTIR antibodies with stabilized binding domains. For the CD40 reporter assay, serial dilutions of ADAPTIR containing wild-type or FcγR-null Fc were combined with human CD40 / NFκB HEK293 luciferase reporter cells (BPS) and CD64-expressing (A) or parental (B) CHOK1SV target cells. After 6 hours, Bio-Glo luciferase reagent (Promega) was added, and luminescence was read on a MicroBeta 2450 Microplate Counter (Perkin Elmer). The y-axis displays light units relative to background signal. (See Example 34.) [Figure 21]This demonstrates the in vivo functionality of anti-PD-L1 and anti-CD40 ADAPTIR antibodies with stabilized binding domains in tumor targeting and killing. 0.5 million human PD-L1-expressing MC38 tumor cells were injected SC into the right flank of female huCD40 / PD-1 / PD-L1 triple knock-in mice (n=5 / group). Treatment was administered by IP injection at 3 mg / kg on days 0, 2, 7, 10, 14, and 17. The mean tumor volume for each group was plotted + SEM. Mice that reached a tumor endpoint of 1500 mm3 or greater had their last recorded tumor volume used for future time points. (See Example 35.) [Figure 22] Panels A-B show the activity of an optimized anti-PD-L1 x anti-CD40 ADAPTIR bispecific with stabilized binding domains capable of stimulating DCs in vitro. Immature DCs were cultured alone (A) or with SCC152 (ATCC) target cells (B) lentivirally transduced to express PD-L1 and human PD-L1 protein (Vectorbuilder), and titrated with ADAPTIR or a control. After 48 hours, cells were harvested and fluorescently labeled for CD86. (See Example 36.) [Figure 23] Figures A-C show the activity of an optimized anti-PD-L1 x anti-CD40 ADAPTIR bispecific with stabilized binding domains in targeting and killing tumors in vitro. SCC152 cells transduced with human PD-L1 and EBV proteins (Vectorbuilder) were co-cultured with immature DCs and EBV-specific T cells. Supernatants were collected after 48 hours and analyzed for IL-12 (A), IFN-γ (B), and TNF-α (C) using a multiplex magnetic bead cytokine assay (Milliplex). (See Example 37.) [Figure 24]Panels A-B show the activity of an optimized anti-PD-L1 x anti-CD40 ADAPTIR bispecific with a stabilized binding domain for tumor targeting and killing in vitro. SCC152 (ATCC) cells were sequentially transduced with lentivirus to express Nuclight Orange (NLO, Sartorius), human PD-L1, and EBV proteins (Vectorbuilder). For the cytotoxicity assay, M2 macrophages, immature DCs, and EBV-specific T cells were prepared separately prior to the assay. Adherent target SCC152 cells expressing NLO, PD-L1, and EBV proteins were plated 1 day prior to assay setup. Titrated ADAPTIR or control macrophages, DCs, and T cells were then added to the SCC152 target cells on assay day 0. Assay plates were imaged every 8 hours over the course of 6 days using an Incucyte reader (Sartorius). In A, a graphical representation of the number of viable SCC152 tumor cells upon addition of 0.22 nM of test or control molecules over time. After 160 hours, tumor cell numbers were assessed and plotted to demonstrate the ability of ADAPTIR to enhance tumor cell killing by T cells (B). (See Example 38.) DETAILED DESCRIPTION OF THE INVENTION

[0042] Provided herein are bispecific polypeptides that specifically bind to PD-L1 and / or CD40. Bispecific molecules that target PD-L1 and / or CD40 are useful for priming anti-tumor immune responses against cancer cells expressing tumor-associated antigens. The molecules provided herein combine a tumor-targeting arm (e.g., a PD-L1-targeting arm) with an immune system agonist (e.g., via a CD40-targeting arm) to cause tumor-dependent cross-linking and agonism of CD40. The bispecific molecules (as well as the PD-L1-binding domain and CD40-binding domain) provided herein have been engineered to combine specific binding with advantageous manufacturability properties, such as thermal stability, solubility, and resistance to shear stress.

[0043] definition The term "about" when used immediately preceding a numerical value means ± up to 10% of that numerical value. For example, "about 40" means ± up to 10% of 40 (i.e., 36-44), ± up to 10%, ± up to 9%, ± up to 8%, ± up to 7%, ± up to 6%, ± up to 5%, ± up to 4%, ± up to 3%, ± up to 2%, ± up to 1%, ± less than ± up to 1%, or any other value or range of values ​​therebetween.

[0044] As used herein, the term "binding domain" or "binding region" refers to a domain, region, portion, or site of a protein, polypeptide, oligopeptide, peptide, antibody, or binding domain derived from an antibody that retains the ability to specifically recognize and bind to a target molecule, such as an antigen, ligand, receptor, substrate, or inhibitor. Exemplary binding domains include antibodies and antibody-like proteins or domains, antibody heavy and light chain variable regions, and single-chain antibody variable regions (e.g., domain antibodies, sFv, scFv, scFab). In certain embodiments, a binding domain comprises or consists of an antigen-binding site (e.g., comprising variable heavy and variable light chain sequences from an antibody arranged within alternative framework regions (FRs) (e.g., human FRs, optionally containing one or more amino acid substitutions), or three light chain complementarity-determining regions (CDRs) and three heavy chain CDRs). Various assays are known for identifying binding domains of the present disclosure that specifically bind to a particular target, including Western blot, ELISA, phage display library screening, and BIACORE® interaction analysis. In some embodiments, a polypeptide of the present disclosure comprises a binding domain that specifically binds to a target antigen expressed by a target cell. In some embodiments, a polypeptide of the present disclosure comprises a binding domain that specifically binds to a target antigen expressed by an effector cell. In some embodiments, a polypeptide of the present disclosure is a multispecific polypeptide and comprises two or more binding domains.

[0045] The binding domain or a protein containing a binding domain is 5 M-1 Affinity or K equal to or exceeding a A binding domain "specifically binds" to a target if it binds to the target with a K (i.e., the equilibrium association constant for a particular binding interaction, in units of 1 / M), while not binding significantly to other components present in the test sample. Binding domains can be classified as "high affinity" and "low affinity" binding domains. A "high affinity" binding domain has a K a is at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , or at least 10 13 M -1 A "low affinity" binding domain refers to a binding domain with a K a up to 10 7 M -1 , up to 10 6 M -1 , up to 10 5 M -1 Alternatively, affinity refers to the equilibrium dissociation constant (K) of a particular binding interaction, in units of M. d ) (e.g., 10 -5 M~10 -13 or about 500 nM, about 300 nM, about 250 nM, about 200 nM, about 150 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, or about 5 nM). The affinity of binding domain polypeptides and single chain polypeptides according to the present disclosure can be readily determined using conventional techniques (see, e.g., Scatchard et al. (1949) Ann. N.Y. Acad. Sci. 51:660, and U.S. Pat. Nos. 5,283,173, 5,468,614, or equivalents).

[0046] As used herein, " conservative substitution " is recognized in the art as the substitution of one amino acid with another amino acid that has similar properties.Exemplary conservative substitutions are well known in the art (see, for example, PCT Application Publication No. WO97 / 09433, page 10, published March 13, 1997; Lehninger, Biochemistry, Second Edition; Worth Publishers, Inc. NY: NY (1975), pp.71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), p.8).

[0047] As used herein, the term "derivative" refers to the modification of one or more amino acid residues of a peptide by chemical or biological means, with or without an enzyme, for example, by glycosylation, alkylation, acylation, ester formation, or amide formation.

[0048] As used herein, a polypeptide or amino acid sequence "derived from" a designated polypeptide or protein refers to the origin of the polypeptide. In certain embodiments, a polypeptide or amino acid sequence is derived from a particular sequence (sometimes referred to as a "parent" or "parental" sequence) and has essentially the same amino acid sequence as the parent sequence or a portion thereof, where the portion consists of at least about 10-20 amino acids, at least about 20-30 amino acids, at least about 30-50 amino acids, or at least about 50-150 amino acids, or is otherwise identifiable by one of skill in the art as having its origin in the parent sequence. For example, a binding domain (e.g., Fab, F(ab'), Fab', scFv, single-domain antibody (sdAb), etc.) can be derived from an antibody. In some embodiments, the binding domain sequence is derived from an antibody or protein by a computer algorithm or in silico.

[0049] A polypeptide derived from another polypeptide may have one or more mutations or changes relative to the parent polypeptide, e.g., one or more amino acid residues substituted with another amino acid residue or one or more amino acid insertions or deletions. In such embodiments, a polypeptide derived from a parent polypeptide and containing one or more mutations or changes is referred to as a "variant." As used herein, the term "variant(s)" refers to a polynucleotide or polypeptide that has a sequence that differs from that of a reference polynucleotide or polypeptide but retains its essential properties. Generally, a variant polynucleotide or polypeptide sequence is similar overall to, and in many regions identical to, the reference polynucleotide or polypeptide sequence. For example, a variant polynucleotide or polypeptide may have at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity compared to an active portion or full-length reference polynucleotide or polypeptide. A polypeptide may contain a non-naturally occurring amino acid sequence. Such variants necessarily have less than 100% sequence identity or similarity with the parent polypeptide. In one embodiment, the variant has an amino acid sequence that is about 60% to less than 100% identical or similar to the amino acid sequence of the parent polypeptide. In other embodiments, the variant has an amino acid sequence that is about 75% to less than 100%, about 80% to less than 100%, about 85% to less than 100%, about 90% to less than 100%, or about 95% to less than 100% identical or similar to the amino acid sequence of the parent polypeptide.

[0050] As used herein, the term "sequence identity" refers to the relationship between two or more polynucleotide sequences or two or more polypeptide sequences.If a position in one sequence is occupied by the same nucleic acid base or amino acid residue at the corresponding position in the comparison sequence, the sequence is said to be "identical" at that position.The sequence identity percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of identical positions.The number of identical positions is then divided by the total number of positions in the comparison window and multiplied by 100 to obtain the percentage of sequence identity.The percentage of sequence identity is determined by comparing two optimally aligned sequences in the comparison window. A comparison window for polynucleotide sequences can be, for example, at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 or more nucleic acids in length. A comparison window for polypeptide sequences can be, for example, at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300 or more amino acids in length. To optimally align sequences for comparison, portions of the polynucleotide or polypeptide sequence within the comparison window can contain additions or deletions, called gaps, while holding the reference sequence constant. An optimal alignment is one that produces the maximum possible number of "identical" positions between the reference and comparison sequences, even with gaps. The version of the program "BLAST 2 Sequences" available from the National Center for Biotechnology Information as of September 1, 2004, can be used to determine the percentage "sequence identity" between two sequences.This program incorporates BLASTN (for nucleotide sequence comparison) and BLASTP (for polypeptide sequence comparison), which are based on the algorithms of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993). When using "BLAST 2 Sequences," which were the default parameters as of September 1, 2004, any other desired parameters can be used, including, but not limited to, word size (3), open gap penalty (11), extension gap penalty (1), gap dropoff (50), expectation value (10), and matrix options. Two nucleotide or amino acid sequences are considered to have "substantially similar sequence identity" or "substantial sequence identity" if the two sequences have at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with each other.

[0051] As used herein, unless otherwise provided, amino acid residue positions in the variable region of an immunoglobulin molecule are numbered according to either the IMGT standard (Brochet et al., Nucl. Acids Res. (2008) 36, W503-508) or the Kabat numbering rules, or according to the EU nomenclature (Ward et al., 1995 Therap. Immunol. 2:77-94), and amino acid residue positions in the constant region of an immunoglobulin molecule are numbered according to the EU nomenclature (Ward et al., 1995 Therap. Immunol. 2:77-94). thed. Bethesda, MD: Public Health Service, National Institutes of Health (1991)) is an alternative system used to refer to the positions of amino acid residues in the variable regions of immunoglobulin molecules, and is sometimes used herein to refer to the positions of amino acid residues in the variable regions of immunoglobulin molecules.

[0052] As used herein, the term "dimer" refers to a biological entity consisting of two subsets associated with each other through one or more forms of intramolecular forces, including covalent bonds (e.g., disulfide bonds) and other interactions (e.g., electrostatic interactions, salt bridges, hydrogen bonds, and hydrophobic interactions), and that is stable under appropriate conditions (e.g., under physiological conditions, in aqueous solutions suitable for recombinant protein expression, purification, and / or storage, or under non-denaturing and / or non-reducing electrophoresis conditions). As used herein, the term "heterodimer" or "heterodimeric protein" refers to a dimer formed from two different polypeptides. Heterodimers do not include antibodies formed from four polypeptides (i.e., two light chains and two heavy chains). As used herein, the term "homodimer" or "homodimeric protein" refers to a dimer formed from two identical polypeptides.

[0053] "Fc region" or "Fc domain" refers to a polypeptide sequence corresponding to or derived from a portion of a source antibody that can bind to cellular Fc receptors and / or the C1q component of complement, thereby mediating the antibody's effector functions. Fc stands for "crystallizable fragment," i.e., a fragment of an antibody that readily forms protein crystals. Individual protein fragments, originally described by proteolytic digestion, can define the overall general structure of immunoglobulin proteins. As originally defined in the literature, the Fc region is a homodimeric protein comprising two polypeptides associated by disulfide bonds, each containing a hinge region, a CH2 domain, and a CH3 domain. However, more recently, the term has been applied to a single-chain monomeric component consisting of a CH3, a CH2, and at least a portion of a hinge sufficient to form a disulfide-bonded dimer with a second such chain. Thus, depending on the context, the use of the term "Fc region" or "Fc domain" herein refers to either the dimeric form or the individual monomers that associate to form the dimeric protein. For a review of immunoglobulin structure and function, see Putnam, The Plasma Proteins, Vol. V (Academic Press, Inc., 1987), pp. 49-140, and Padlan, Mol. Immunol. 31:169-217, 1994. As used herein, the term Fc includes naturally occurring sequence variants.

[0054] "Immunoglobulin constant region" or "constant region" or "constant domain" is a term defined herein to refer to a peptide or polypeptide sequence corresponding to or derived from part or all of one or more constant domains of an immunoglobulin. In certain embodiments, the constant region comprises IgG CH2 and CH3 domains, e.g., IgG1 CH2 and CH3 domains. In certain embodiments, the constant region does not comprise a CH1 domain. In certain embodiments, the constant domains comprising the constant region are human. In some embodiments, the constant region of the fusion proteins of the present disclosure lacks or has minimal effector function while retaining the ability to bind to some Fc receptors, such as fetal Fc receptor (FcRn), and retaining a relatively long in vivo half-life. For example, the constant region of the fusion proteins of the present disclosure does not confer or substantially reduces antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement activation, and / or complement-dependent cytotoxicity (CDC). In other variations, the fusion proteins of the present disclosure comprise a constant domain that retains one or more effector functions, e.g., ADCC and / or CDC. In certain embodiments, the binding domain of the present disclosure is fused to a human IgG1 constant region, where the IgG1 constant region is mutated at one or more of the following amino acids: E233, L234, L235, G236, G237, E318, K320, K322, or any combination thereof (EU numbering). For example, any one or more of these amino acids can be changed to alanine, valine, or proline. In some embodiments, the IgG1 constant region has one or more of the following amino acid mutations: E233P, L234A, L234V, L235A, G237A, E318A, K320A, K322A. In some embodiments, the IgG1 Fc domain is mutated at each of E233, L234, L235, G236, G237, E318, K320, K322 (EU numbering). In some embodiments, the IgG1 constant region is deleted of one or more of the following amino acids: E233, L234, L235, G236, G237, E318, K320, K322 (EU numbering).In some embodiments, the IgG1 constant region has each of the following deleted: E233, L234, L235, G236, G237, E318, K320, K322 (EU numbering).

[0055] "Light chain variable region" ("light chain variable domain" or "V L ") and a "heavy chain variable region" (also called a "heavy chain variable domain" or "V H The terms "CL" and "CL" refer to the variable binding regions from antibody light and heavy chains, respectively. The variable binding regions are composed of distinct, well-defined subregions known as "complementarity-determining regions" (CDRs) and "framework regions" (FRs). In some embodiments, the FRs are humanized. The term "CL" refers to the "immunoglobulin light chain constant region" or "light chain constant region," i.e., the constant region from an antibody light chain. The term "CH" refers to the "immunoglobulin heavy chain constant region" or "heavy chain constant region," which can be further divided into CH1, CH2, and CH3 domains (IgA, IgD, IgG) or CH1, CH2, CH3, and CH4 domains (IgE, IgM) depending on the antibody isotype. "Fab" (Fragment Antigen Binding) is the portion of an antibody that binds to an antigen and comprises the variable region and CH1 domain of the heavy chain linked to the light chain via an interchain disulfide bond.

[0056] As used herein, the term "linker" generally refers to a short polypeptide sequence connecting two subdomains of a polypeptide. Non-limiting examples of linkers include flexible linkers containing glycine-serine repeats and linkers derived from (a) the interdomain region of a transmembrane protein (e.g., a type I transmembrane protein), (b) the stalk region of a type II C-lectin, or (c) an immunoglobulin hinge. In some embodiments, the linker provides a spacer function that is compatible with the interaction of the two sub-binding domains so that the resulting polypeptide retains the same specific binding affinity for the target molecule as an antibody comprising the same light and heavy chain variable regions. In certain embodiments, the linker is composed of 5 to about 35 amino acids, e.g., about 15 to about 25 amino acids. As used herein, the phrase "linker between CH3 and CH1 or CL" refers to one or more amino acid residues (e.g., about 2-12, about 2-10, about 4-10, about 5-10, about 6-10, about 7-10, about 8-10, about 9-10, about 8-12, about 9-12, or about 10-12) between the C-terminus of the CH3 domain (e.g., wild-type CH3 or mutant CH3) and the N-terminus of the CH1 domain or CL domain (e.g., Cκ).

[0057] In some embodiments, depending on the context, the linker may be: (1) a V in a single-chain Fv (scFv); H Area and V LThe term "Fc binding domain linker" may refer to (1) the polypeptide region between a first binding domain and a second binding domain in a multispecific polypeptide comprising two binding domains, or (2) the polypeptide region between a first binding domain and a second binding domain in a multispecific polypeptide comprising two binding domains. In the latter example, when a linker connects two or more binding domains, such a linker is referred to herein as an "Fc binding domain linker." In some embodiments, an Fc binding domain linker may directly link or connect two or more binding domains, resulting in a construct comprising the following structure: binding domain-Fc binding domain linker-binding domain. In some embodiments, a multispecific polypeptide described herein comprises, in order from amino terminus to carboxyl terminus, (i) a first binding domain, (ii) an Fc binding domain linker, and (iii) a second binding domain. In some embodiments, a multispecific polypeptide comprises, in order from amino terminus to carboxyl terminus, (i) a second binding domain, (ii) an Fc binding domain linker, and (iii) a first binding domain. In some embodiments, an Fc binding domain linker may link or connect two or more binding domains by linking at least one binding domain to a non-binding domain polypeptide, such as an immunoglobulin Fc domain (i.e., a polypeptide comprising the structure: Ig hinge-Ig constant region). In such embodiments, the resulting construct may comprise the following structure: binding domain-Fc domain-Fc binding domain linker-binding domain. In some embodiments, the multispecific polypeptides described herein comprise, in order from amino terminus to carboxyl terminus, (i) a first binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) an Fc binding domain linker, and (v) a second binding domain. In some embodiments, the multispecific polypeptides comprise, in order from amino terminus to carboxyl terminus, (i) a second binding domain, (ii) an Fc binding domain linker, (iii) an immunoglobulin constant region, (iv) a hinge region, and (v) a first binding domain.The polypeptide region between the immunoglobulin constant region and the second binding domain in a multispecific polypeptide comprising two binding domains (e.g., an Fc binding domain linker) may also be referred to as a "carboxyl-terminal linker" or an "amino-terminal linker", depending on the orientation of the domains within the multispecific polypeptide.

[0058] In some embodiments, "hinge" or "hinge region" refers to a polypeptide derived from an immunoglobulin hinge region and located between the binding domain and the immunoglobulin constant region in a polypeptide described herein. A "wild-type immunoglobulin hinge region" refers to the naturally occurring upper and middle hinge amino acid sequences inserted between and connecting the CH1 and CH2 domains (in the case of IgG, IgA, and IgD) or between and connecting the CH1 and CH3 domains (in the case of IgE and IgM) found in the heavy chain of an antibody. In certain embodiments, the wild-type immunoglobulin hinge region sequence is human and may comprise a human IgG hinge region (e.g., an IgG1, IgG2, IgG3, or IgG4 hinge region).

[0059] An "altered immunoglobulin hinge region" or "variant immunoglobulin hinge region" refers to a hinge region polypeptide having one or more mutations, substitutions, insertions, or deletions compared to a corresponding parent wild-type immunoglobulin hinge region. In some embodiments, the altered immunoglobulin hinge region is at least about 70% identical (at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identical) to a wild-type immunoglobulin hinge region. In certain embodiments, the altered immunoglobulin hinge region is a fragment of a wild-type immunoglobulin hinge region having a length of about 5 amino acids (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids) up to about 120 amino acids (e.g., about 10 to about 40 amino acids, or about 15 to about 30 amino acids, or about 15 to about 20 amino acids, or about 20 to about 25 amino acids). Typically, an altered immunoglobulin hinge region that is a fragment of a wild-type immunoglobulin hinge region comprises an IgG core hinge region (e.g., a polypeptide comprising the sequence CXXC, where X is any amino acid) as disclosed in U.S. Patent Application Publication Nos. 2013 / 0129723 and 2013 / 0095097.

[0060] As used herein, the term "humanization" refers to the process of using genetic engineering techniques to render antibodies or immunoglobulin-binding proteins and polypeptides derived from non-human species (e.g., mouse or rat) less immunogenic to humans while retaining the antigen-binding properties of the original antibody. In some embodiments, the binding domain(s) of the antibodies or immunoglobulin-binding proteins and polypeptides (e.g., light and heavy chain variable regions, Fab, scFv) are humanized. Non-human binding domains can be engineered using a technique known as CDR grafting (Jones et al., Nature 321:522 (1986)), as well as "reshaping" (Verhoeyen, et al., 1988 Science 239:1534-1536; Riechmann, et al., 1988 Nature 332:323-337; Tempest, et al., Bio / Technol 1991 9:266-271), "hyperchimerization" (Queen, et al., 1989 Proc Natl Acad Sci USA 86:10029-10033; Co, et al., 1991 Proc Natl Acad Sci USA 88:2869-2873; Co, et al., 1992 J Immunol 148:1149-1154), and "veneering" (Mark, et al. al., "Derivation of therapeutically active humanized and veneered anti-CD18 antibodies." In: Metcalf BW, Dalton BJ, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994:291-312), and variations thereof. Other regions of antibodies or immunoglobulin-binding proteins and polypeptides, such as hinge and constant region domains, may also be humanized if derived from non-human sources. Knowledge in the art regarding humanized antibodies is applicable to polypeptides according to the present disclosure, even if these polypeptides are not antibodies.

[0061] As used herein, the term "patient in need" or "subject in need" refers to a patient or subject at risk for or suffering from a disease, disorder, or condition suitable for treatment or amelioration with a binding protein or multispecific polypeptide, or composition thereof, provided herein. The terms "subject" and "patient" are used interchangeably.

[0062] As used herein, the term "pharmaceutically acceptable" refers to molecular entities and compositions that generally do not produce allergic or other serious adverse reactions when administered using routes well known in the art. Molecular entities and compositions that are approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans, are considered "pharmaceutically acceptable."

[0063] As used herein, the term "promoter" refers to a DNA region involved in binding RNA polymerase and initiating transcription.

[0064] As used herein, the terms "nucleic acid," "nucleic acid molecule," or "polynucleotide" refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, these terms encompass nucleic acids containing analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses not only the sequence explicitly indicated, but also conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al. (1985) J. Biol. Chem. 260:2605-2608; Cassol et al. (1992); Rossolini et al. (1994) Mol. Cell. Probes 8:91-98). The term nucleic acid is used synonymously with gene, cDNA, and mRNA encoded by a gene. As used herein, the terms "nucleic acid," "nucleic acid molecule," or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA generated using nucleotide analogs, and derivatives, fragments, and homologs thereof.

[0065] The term "expression" refers to the biosynthesis of a product encoded by a nucleic acid. For example, in the case of a nucleic acid segment encoding a polypeptide of interest, expression involves transcription of the nucleic acid segment into mRNA and the translation of mRNA into one or more polypeptides.

[0066] The terms "expression unit" and "expression cassette" are used interchangeably herein to refer to a nucleic acid segment that encodes a polypeptide of interest and is capable of providing expression of the nucleic acid segment in a host cell. An expression unit typically includes a transcription promoter, an open reading frame encoding the polypeptide of interest, and a transcription terminator, all in an operable configuration. In addition to a transcription promoter and terminator, an expression unit may further include other nucleic acid segments, such as, for example, an enhancer or a polyadenylation signal.

[0067] As used herein, the term "expression vector" refers to a linear or circular nucleic acid molecule containing one or more expression units. In addition to one or more expression units, an expression vector may also contain additional nucleic acid segments, such as one or more replication origins or one or more selection markers. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.

[0068] As used herein, "polypeptide," "polypeptide chain," or "protein" refers to a single, linear, and contiguous arrangement of covalently bonded amino acids. A polypeptide may form one or more intrachain disulfide bonds. With respect to the polypeptides described herein, reference to modifications or changes of amino acid residues corresponding to those identified by SEQ ID NOs includes post-translational modifications of such residues. The terms polypeptide and protein also encompass embodiments in which two polypeptide chains are linked in a nonlinear manner, e.g., via interchain disulfide bonds. For example, a native immunoglobulin molecule is composed of two heavy chain polypeptides and two light chain polypeptides. Each heavy chain polypeptide associates with a light chain polypeptide through an interchain disulfide bond between the heavy and light chain polypeptides to form two heterodimeric proteins or polypeptides (i.e., a protein composed of two heterologous polypeptide chains). The two heterodimeric proteins then associate through an additional interchain disulfide bond between the heavy chain polypeptides to form an immunoglobulin protein or polypeptide. As used herein, a protein or polypeptide may be an antibody or an antigen-binding fragment of an antibody.

[0069] As used herein, a "PD-L1 binding protein" may be used synonymously with a "PD-L1 binding polypeptide." Such molecules specifically bind to the programmed death-ligand 1 protein (PD-L1) (e.g., human PD-L1).

[0070] As used herein, "CD40 binding protein" may be used synonymously with "CD40 binding polypeptide." Such molecules specifically bind to cluster of differentiation 40 protein (CD40) (e.g., human CD40).

[0071] As will be understood by those skilled in the art, proteins and polypeptides are defined herein in terms of the amino acid sequences of the individual polypeptide chains, which amino acid sequences are indicated by the SEQ ID NOs referenced throughout this disclosure. Polypeptides and proteins may also include non-peptidic components, such as carbohydrate groups. Carbohydrate and other non-peptidic substituents may be added to proteins or polypeptides by the cell in which the protein is produced and will vary depending on the type of cell. Proteins and polypeptides are defined herein in terms of their amino acid backbone structures. Substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.

[0072] The terms "amino-terminal" and "carboxyl-terminal" are used herein to denote positions within a polypeptide. Where the context permits, these terms are used with respect to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a particular sequence located at the carboxyl terminus of a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.

[0073] As used herein, the terms "transformation," "transfect," and "transduction" refer to the introduction of a nucleic acid (i.e., a nucleotide polymer) into a cell. As used herein, the term "genetic transformation" refers to the introduction and incorporation of DNA, particularly recombinant DNA, into a cell. The introduced nucleic acid may be introduced into the cell via an expression vector.

[0074] As used herein, "antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcgR (e.g., mononuclear cells such as natural killer (NK) cells and macrophages) recognize bound antibodies (or other proteins capable of binding FcgR) on target cells, subsequently causing lysis of the target cells. In principle, any effector cell bearing an activated FcgR can be induced to mediate ADCC. The primary cells for mediating ADCC are NK cells, which express only FcgRIII, whereas monocytes can express FcgRI, FcgRII, and FcgRIII, depending on their state of activation, localization, or differentiation. For a review of FcgR expression on hematopoietic cells, see, e.g., Ravetch et al., 1991, Annu. Rev. Immunol., 9:457-92.

[0075] As used herein with respect to a polypeptide or protein, the term "having ADCC activity" means that a polypeptide or protein, for example, one comprising an Fc domain (e.g., an immunoglobulin constant region having an immunoglobulin hinge region and CH2 and CH3 domains) derived from an IgG (e.g., IgG1), is capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) through binding of a cytolytic Fc receptor (e.g., FcgRIII) on cytolytic immune effector cells expressing the Fc receptor (e.g., NK cells). In some embodiments, a multispecific polypeptide or protein comprising an Fc domain may lack effector function (e.g., null ADCC activity) as a result of mutations in the CH2 and / or CH3 domains.

[0076] As used herein, "complement-dependent cytotoxicity" and "CDC" refer to the process by which components in normal serum ("complement"), along with antibodies or other C1q complement-binding proteins, bind to target antigens and cause lysis of target cells expressing the target antigen. Complement consists of a group of serum proteins that act in concert and in an orderly sequence to exert their effect.

[0077] As used herein, the terms "classical complement pathway" and "classical complement system" are synonymous and refer to a specific pathway for complement activation. The classical pathway requires an antigen-antibody complex for initiation and involves the ordered activation of nine major protein components, designated C1 through C9. At several steps in the activation process, the product is an enzyme that catalyzes a subsequent step. This cascade results in the amplification and activation of a large amount of complement with a relatively small initial signal.

[0078] As used herein with respect to a polypeptide or protein, the term "having CDC activity" means that a polypeptide or protein, such as one comprising an Fc domain (e.g., an immunoglobulin constant region having an immunoglobulin hinge region and CH2 and CH3 domains) from an IgG (e.g., IgG1), can mediate complement-dependent cytotoxicity (CDC) through binding of the C1q complement protein and activation of the classical complement system. In some embodiments, a multispecific polypeptide or protein may lack effector function (e.g., null CDC activity) as a result of one or more mutations in the CH2 and / or CH3 domains.

[0079] As used herein, the term "effector cell" refers to a cell of the immune system that can lyse or kill target cells, such as tumor cells. As used herein, effector cells may refer to lymphocytes, such as T cells, natural killer (NK) cells, or NKT cells, monocytes, macrophages, dendritic cells, or granulocytes. In certain embodiments, the term effector cell refers to a T cell, an NK cell, or an NKT cell.

[0080] As used herein, the terms "treatment," "treating," or "ameliorating" refer to therapeutic treatment. Treatment is therapeutic if at least one symptom of the disease in the individual receiving treatment is improved, or if the treatment can slow the progression of the individual's disease or prevent the onset of further related diseases or symptoms.

[0081] As used herein, the term "therapeutically effective amount (or dose)" or "effective amount (or dose)" of a polypeptide or protein, or composition thereof, described herein, refers to an amount of compound sufficient to result in a statistically significant improvement in one or more symptoms of the disease being treated, or a statistically significant improvement in organ function. When referring to an individual active ingredient administered alone, the therapeutically effective dose refers to that ingredient alone. When referring to a combination, the therapeutically effective dose refers to the amount of the combination of active ingredients that results in a therapeutic effect, whether administered sequentially or simultaneously (either in the same formulation or simultaneously in separate formulations).

[0082] As used herein, a "multispecific polypeptide" refers to a polypeptide comprising two or more binding domains, each capable of specifically binding to a target antigen. For example, a polypeptide described herein may comprise two, three, four, or more binding domains and may bind to two, three, four, or more target antigens. In some embodiments, a multispecific polypeptide is a bispecific polypeptide. As used herein, a "bispecific polypeptide" comprises two binding domains and is capable of binding to two distinct target antigens. In some embodiments, a bispecific polypeptide described herein comprises a first binding domain that specifically binds to a cell surface antigen expressed on a target cell. In some embodiments, a bispecific polypeptide described herein comprises a binding domain that specifically binds to a cell surface antigen expressed on an effector cell. In certain embodiments, a multispecific polypeptide is an ADAPTIR homodimeric bispecific polypeptide in the scFv-Fc-scFv format.

[0083] Scaffold-based multispecific polypeptides are described, for example, in PCT Publication Nos. WO2007 / 146968, WO2010 / 040105, WO2010 / 003108, WO2016 / 094873, WO2017 / 053469, U.S. Patent Application Publication No. 2006 / 0051844, and U.S. Patent Nos. 7,166,707 and 8,409,577, each of which is incorporated herein by reference in its entirety. In certain embodiments, the multispecific polypeptides described herein are bispecific polypeptides and may comprise an scFv-Fc-scFv structure, also referred to herein as an ADAPTIR polypeptide. Polypeptides comprising such a structure comprise, from N- to C-terminus, a first scFv binding domain, a hinge region, an immunoglobulin constant region, and a second scFv binding domain. In some embodiments, the structure of the polypeptide comprises, from N-terminus to C-terminus, a first scFv binding domain, a hinge region, an immunoglobulin constant region, an Fc binding domain linker, and a second scFv binding domain.

[0084] Bispecific Binding Polypeptides Provided herein are bispecific binding polypeptides having a first binding domain and a second binding domain, where the first binding domain binds to a tumor-associated antigen and the second binding domain binds to an immune system agonist. The bispecific binding polypeptide may have an scFv-Fc-scFv structure. In some embodiments, the tumor-associated antigen is PD-L1. In some embodiments, the immune system agonist is CD40. In some embodiments, the bispecific binding polypeptide binds to PD-L1 and CD40. In some embodiments, the bispecific binding molecule binds to PD-L1 and a protein selected from 4-1BB, CD3, and OX40. In some embodiments, the bispecific binding molecule binds to CD40 and a tumor-associated antigen selected from ROR1 and EGFR.

[0085] Provided herein are bispecific binding polypeptides that bind to PD-L1 and CD40 and comprise the sequence of molecules PC401003, PC401119, PC401120, PC401122, PC401124, PC401128, PC401129, PC401132, PC401133, PC401144, or PC401145 as shown in Table 1. Also provided herein are bispecific binding polypeptides that bind to PD-L1 and CD40 and comprise the sequence of the set of 12 CDR sequences of any one of molecules PC401003, PC401119, PC401120, PC401122, PC401124, PC401128, PC401129, PC401132, PC401133, PC401144, and PC401145. Further provided herein are bispecific binding polypeptides that bind to PD-L1 and CD40 and comprise the sequences of the set of four variable region sequences of any one of molecules PC401003, PC401119, PC401120, PC401122, PC401124, PC401128, PC401129, PC401132, PC401133, PC401144, and PC401145.

[0086] In Table 1, CDR sequences (Kabat definition) are single underlined when shown as part of a longer sequence. In these scFv sequences, the scFv linker sequence is in italics, the VL sequence is in bold, and the VH sequence is in regular font. In the bispecific molecule sequences, the PD-L1-binding domain sequence is in bold, the CD40-binding domain sequence is in italics, the Fc sequence is in regular font, and the H125 linker is double underlined. Amino acid substitutions are compared to the corresponding CD4001242 sequence in the PC401119, PC401120, PC401122, PC401124, PC401128, PC401129, PC401132, PC401133, PC401144, and PC401145 molecules. The humanized 1D5 anti-CD40 sequence is shown in bold. Table 1. Exemplary amino acid sequences of bispecific molecules and their components [Table 1] JPEG2026501724000003.jpg215159JPEG2026501724000004.jpg226159JPEG2026501724 000005.jpg218159JPEG2026501724000006.jpg222159JPEG2026501724000007.jpg22115 9JPEG2026501724000008.jpg219159JPEG2026501724000009.jpg224159JPEG2026501724 000010.jpg222159JPEG2026501724000011.jpg224159JPEG2026501724000012.jpg44159

[0087] Provided herein is a PD-L1-binding polypeptide that specifically binds to human PD-L1, comprising, in amino-terminal to carboxyl-terminal or carboxyl-terminal to amino-terminal order: (a) a first binding domain, (b) a hinge region, (c) an immunoglobulin constant region, and (d) a second binding domain, wherein the first binding domain is a PD-L1-binding domain and the second binding domain binds to an immunostimulatory protein, or the first binding domain binds to an immunostimulatory protein and the second binding domain is a PD-L1-binding domain. The first binding domain, the second binding domain, or both binding domains can be scFvs. In some embodiments, the immunostimulatory protein is CD40, 4-1BB, CD3, or OX40.

[0088] In some embodiments, the PD-L1 binding domain comprises (i) an immunoglobulin heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3, and (ii) an immunoglobulin light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3. In some embodiments, HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 5, LCDR2 comprises SEQ ID NO: 6, and LCDR3 comprises SEQ ID NO: 7. In some embodiments, the VH comprises SEQ ID NO: 4 and the VL comprises SEQ ID NO: 8.

[0089] In some embodiments, the PD-L1 binding domain comprises SEQ ID NO: 9. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 9.

[0090] In some embodiments, the second binding domain comprises (i) an immunoglobulin heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3, and (ii) an immunoglobulin light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3. Table 2. Sequence Listing >PDL01001-SEQ ID NO:53 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHEL TCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERSSSLNDIFEAQKIEWHEDYKDDDDKDYKDDDDKDYKDDDDKHHHHHHHHHH >PDL01002-SEQ ID NO:54 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNA PYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERSSSEPRGP TIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIE RTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK >PDL01003-SEQ ID NO: 55 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLTSLIVYWEMEDKNIIQFVHGEEDLKVQHSNYRQRAQLLKDQLSLGNAALRITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHEL TCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLLNVTSTLRINTTANEIFYCIFRRLDPEENHTAELVIPELPLALPPNERSSSLNDIFEAQKIEWHEDYKDDDDKDYKDDDDKDYKDDDDKHHHHHHHHHH >PDL01004-SEQ ID NO:56 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLTSLIVYWEMEDKNIIQFVHGEEDLKVQHSNYRQRAQLLKDQLSLGNAALRITDVKLQDAGVYRCMISYGGADYKRITVKVNA PYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLLNVTSTLRINTTANEIFYCIFRRLDPEENHTAELVIPELPLALPPNERSSSEPRGP TIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIE RTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK >PDL01011-SEQ ID NO:57 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYRKINQRISVDPATSEHELI CQAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGMLLNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRSSSLNDIFEAQKIEWHEDYKDDDDKDYKDDDDKDYKDDDDKHHHHHHHHHH >PDL01017-SEQ ID NO:58 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVN APYRKINQRISVDPATSEHELICQAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGMLLNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRSSSEPRGP TIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIE RTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK >PDL01021 SEQ ID NO: 59 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELT CQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET >PDL01022-SEQ ID NO: 60 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLTSLIVYWEMEDKNIIQFVHGEEDLKVQHSNYRQRAQLLKDQLSLGNAALRITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELT CQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLLNVTSTLRINTTANEIFYCIFRRLDPEENHTAELVIPELPLALPPNERTHLVILGAIFLLLGVALTFIFYLRKGRMMDMKKCGIRVTNSKKQRDTQLEET >PDL01026-SEQ ID NO: 61 FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAH PSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL >PDL01031_2C11-SEQ ID NO: 62 DVKLVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGNYTYYPDSVKGRFTISRDNAKNTLYLQLSSLKSEDTAMYYCTRDQAYYGNLFTYWGQGTLVTVSAGGGG SGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTITCRASENIYSFLAWYQQKQGKSPQLLVYKAKTLTEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGSPPETFGGGTKVEIK >PDL01032_3G5-SEQ ID NO: 63 QIQLQQSGPELVKPGASVKISKASGYTFTDYYINWVKQKPEQGLEWIGWTFPGSINTKYNEKFKGKATLTVDTSSSTAYMQLNSLTSEDTAVYFCARSPYEFDYWGQGTTLTVSSGGGGS GGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCSASQGIRNYLNWYQQKPDGTVKLLIYYTSTLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPWTFGGGTRLEIK >PDL01033_3G11-SEQ ID NO: 64 EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGKKLEYMGYISYTGSTYYNPSLKSRISIARDTSKNQYFLQLNSVTTEDTATYYCTRGENWDGGYFDYWGQGTTLTVSSGGGG SGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCSASQGIRNYLNWYQQKPDGTVKLLIYYTSTLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPWTFGGGTRLEIK >PDL01034_5F9-SEQ ID NO: 65 EVKLVESGGGLVQPGGSRKLSCAASGFTFSDYGMAWVRQAPGKGPEWVAFITSLAYIIFYSDTVTGRFTISRENAKNTLYLEMSSLRSEDTAVYYCARNYGSSPYYFDYWGQGTTLTVSSGGG GSGGGGSGGGGSGGGGSDIQMTQSPSSLSTSLGGKVTITCKASQDINKFISWYQHKPGKGPRLLIHYTSSLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLYTFGGGTKLEIK >5F9_HCDR1-DYGMA (SEQ ID NO: 1) >5F9_HCDR2-FITSLAYIIFYSDTVTG (SEQ ID NO: 66) >5F9_HCDR3-NYGSSPYYFDY (SEQ ID NO: 3) >5F9_LCDR1-KASQDINKFIS (SEQ ID NO: 67) >5F9_LCDR2-YTSSLQP (SEQ ID NO: 68) >5F9_LCDR3-LQYDNLYT (SEQ ID NO: 7) >PDL01034_5F9_VH-SEQ ID NO: 276 DIQMTQSPSSLSTSLGGKVTITCKASQDINKFISWYQHKPGKGPRLLIHYTSSLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLYTFGGGTKLEIK >PDL01034_5F9_VL-SEQ ID NO: 277 EVKLVESGGGLVQPGGSRKLSCAASGFTFSDYGMAWVRQAPGKGPEWVAFITSLAYIIFYSDTVTGRFTISRENAKNTLYLEMSSLRSEDTAVYYCARNYGSSPYYFDYWGQGTTLTVSS >PDL01035_5F11-SEQ ID NO: 69 EVKFEESGGGLVQPGGSMKLSCFASGFTFSYYWMNWVRQSPEKGLEWIAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLEMNNLRAEDTGIYHCTRPPIYYGNYEAYWGQGTLVTVSAGG GGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASLGKVTITCKASQDINKYIAWYQHKPGKGPRLLIHYTSILQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLRTFGGGTKLEIK >PDL01036_6F6-SEQ ID NO: 70 QGQMQQSGAELVKPGASVKLSCKTSGFTFSSSYITWLKQKPGQSLEWIAWIYVGTGGISYNQKFTGKAQLTVDTSSSTAYMQFSSLTTEDSAIYYCARHGRYHWYFDVWGAGTTVTVSSGGGG SGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVTITCKASQDVGTAVAWYQKKPGQSPKLLISWASTRHTGVPDRFTGSGSGTFTLTISNVQSEDLADYFCQQYSSYPYTFGGGTNLEIK >PDL01037_7A3-1-SEQ ID NO: 71 EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNRLEYMGFRSYSGTTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCARFSDFGSSENTMDYWGQGTSVTVSSGGG GSGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVTITCKASQDVGTAVAWYQKKPGQSPKLLISWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGGGTNLEIK >PDL01038_7A3-2-SEQ ID NO: 72 EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNRLEYMGFRSYSGTTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCARFSDFGSSENTMDYWGQGTSVTVSSGGG GSGGGGSGGGGSGGGGSDVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPFTFGAGTKLEIK >PDL01039_7H11-SEQ ID NO: 73 EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGKKLEYMGYISYTGSTYYNPSLKSRISIARDTSKNQYFLQLNSVTTEDTATYYCTRGENWDGGYFDYWGQGTTLTVSSGGGGSG GGGSGGGGSGGGGSDIVLTQSPASLAVSLGQRATISCRASKSVSKSGYSYLHWYQQKPGQTPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSWELPYTFGGGTKLEIK >PDL01040_10B2-SEQ ID NO: 74 KVQLQQSGAEVVKPGASVKLSCKASGYNLTEYIIHWVKQRSGQGLEWIGWFYPGSGSIKYNEKFKDKATLTADKSSTTVYMDLSRLTSEDSAVYFCARHGLYYGFPYWGQGTSVTVSSGGGG SGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLQSGVPSRFSGSGSGTDYSLTITTLEQEDFATYFCQQGNMFPYTFGGGTKLEIK >PDL01060-SEQ ID NO: 75 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMNWVRQAPGKGPEWVASITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSS GGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQHKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGRDYTLTISSLQPEDFATYYCLQYDNLYTFGQG TKLEIKSSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >PDL01065-SEQ ID NO: 76 EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYGMSWVRQAPGKGPEWVAAITSLAYIIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSS GGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQHKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGRDYTLTISSLQPEDFATYYCLQYDNLYTFGQG TKLEIKSSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >PDL01085-SEQ ID NO: 77 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIKSSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Humanized 5F9_VH_DNA - Accession number 78 GAGGTACAACTGGTAGAATCAGGGGGCGGACTGGTACAGCCCGGGGGCTCCCTGAGACTGAGTTGCGCCGCCTCTGGCTTTACTTTCTCCGATTACGGTATGGCATGGGTTAGACAAGCCCCCGGAAAAGGGCTCGAATGGGTGTCATTCATCACATCACTGGCCTACATTATCTATTATGCCGACTCCGTTAAGGGGCGGTTCACCATTAGCCGTGACAACGCAAAGAATAGTCTTTACCTGCAAATGAACTCTCTCAGGGCAGAAGATACAGCCGTCTATTACTGTGCCAGAAACTATGGTAGTTCTCCTTATTACTTTGATTACTGGGGACAAGGAACTTTGGTTACCGTGAGCTCA >Humanized 5F9_VL_DNA - Accession number 79 GATATTCAGATGACCCAAAGTCCATCATCCCTTAGTGCCTCCGTGGGGGATAGAGTAACTATCACATGCCGCGCTAGTCAAGATATAACAAATTTTTGTCCTGGTATCAGCAGAAGCCTGGTAAAGCTCCAAATTGCTGATCCATTATACCTCTTCCCTGCAATCTGGGGTACCTTCCCGTTTCTCTGGGTCCGGATCAGGTACAGACTTCACTCTTACCATAAGCTCACTCCAACCAGAAGATTTCGCAACCTATTACTGTCTCCAGTACGATAACCTGTATACTTTTGCCAGGAACTAAGTTGGAGATTAAG >CD4001003- அக்குக்க்கியுக்க்கு80 EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETRCHQHKYCDPNGLLRVQQKGTSETDTICTCEEGLHCTSESCESVCPHRSCLPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCRPWTSCETKDLVVQQAGTNKTDVVCGPQDRQRSSSEPRGPTIKPCPPKCPAPNLLGGPSVFI FPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFCKKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKFSRTPGK >CD4001004- அக்க்கியுக்க்கு 81 EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETRCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGLHCTSESCVPHRSCLPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCRPWTSCETKDLVVQQAGTNKTDVVCGPQDRQRHHHHHHHHHHGLNDIFEAQKIEWHEEPEA >CD4001005-SEQ ID NO:82 EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLSSSEPRGPTIKPCPPCKCPAPNLLGGPSVFIF PPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRA PQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK >CD4001006-SEQ ID NO:83 EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLHHHHHHHHHHGLNDIFEAQKIEWHEEPEA >CD4001012-SEQ ID NO:84 EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPV GFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ >CD4001013-SEQ ID NO:85 EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETRCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGLHCTSESCESCVPHRSCLPGFGVKQIATGVSDTICEPCPVG FFSNVSSAFEKCRPWTSCETKDLVVQQAGTNKTDVVCGPQDRQRALVVIPICLGILFVILLLVLVFIKKVAKKPNDKVPHPKQEPQEINFPDDLPGSNPAAPVQETLHGCQPVTQEDGKESRISVQERQ >CD4001014-SEQ ID NO:86 EVQLQQSGPELVKPGASMKISKASGYSITGYTMNWVKQSHGKNLEWIGLINTYTGGTTYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCAGTGTGAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSQIVLTQSPAIMSSSPGEKVTMTCSASSSVSYIHWYQQKSGTSPK RWIYDTSKLASGVPARFSGSGTSYSLTINSVEAEDAATYYCQQWRTNPYTFGGGTKLEIKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSV KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001015-SEQ ID NO:88 DVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEWVAYISSGSNTIYYADTLKGRFTISRDNPRNTLFLQMTSLRSEDTAIYYCTRRPIPGSMDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSQIVLTQSPALISAFPGEKVTMTCSASSSVTYMHWYQQKPRSSP KSWIYLTSNLASGVPTRFSGSGSGTSYSLTISSMEAEDAATYFCQQWSTNSLTFGAGTKLEIKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSV KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001016-SEQ ID NO: 90 QVQLQQSGAELMNPGASVRISCKATGYTFSTSWIEWIKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTADTSSNTAYMQLSSLTFEDSAVYYCARGDDGSYVRWYFDVWGAGTTVTVSSGGGGSGGGGSGGGSGGGGSDIVMSQSPSSLAVSVGEKVTMSCKSSQSLLFSVNQKNYLAWY QQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTIGSVKAEDLAVYYCQHYYSYPWTFGGGTKLEIKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYA DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001017-SEQ ID NO:92 DVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEWVAYISSGSNTIYYADTLKGRFTISRDNPRNTLFLQMTSLRSEDTAIYYCTRRPIPGAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSQIVLTQSPALISAFPGEKVTMTCSASSSVTYMHWYQQKPRSSP KSWIYLTSNLASGVPSRFSGSGSGTSYSLTISSMEAEDAATYFCQQWSTNSLTFGAGTKLEIKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSV KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001018-SEQ ID NO:94 EVMLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPERRLEWVASISSAGTYIYYPDSVKGRFTISRDNAKNILYLQMNSLRSEDTAMYYCARHGNGYDPLWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHYNGYTYLHWYL QKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQTTHVPLTFGAGTKLELREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYA DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001019-SEQ ID NO:96 DVQLVESGGGLVQPGGSRKLSCAASGFAFSSFGIHWVRQSPEKGLEWVAYISGGSSTIYYADSLKGRFTISRDNPKNTLFLQMTSLRSEDTAIYYCVRRPIPGAMDYWGQGISVTVSSGGGGSGGGGSGGGGSGGGGSQIVLTQSPALMSASPGEKVTMTCRASSRVSYIHWYQQKPRSSP KSWIYLTSNLASGVPSRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNSLTFGAGTKLELKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSV KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001020-SEQ ID NO:98 DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSITIYYADTLKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCTRRPIPGAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSQIVLTQSPALMSASPGEKVTMTCSASSSVTYMHWYQQKPRSSP KSWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSDSLTFGAGTKLELKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSV KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001021-SEQ ID NO: 100 EVLLQQSGPELVKPGASVKISCKASGYKFNDYNIDWVKQSQGKSLEWIGNINPDNGGTIYNQKFKGKATLTVDKSSSTPYMELRSLTSEDTAVYFCARERDNRYDRWSAYWGQGTLVTVSAGGGGSGGGGSGGGGGSGGGGSDIVMTQSQKFMSTSVGDRVSVTCKASQNVGANVAWYQQKPG QSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVHSDDLAEYFCQQYNTYPFTFGSGTKLEIKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADS VKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001022-SEQ ID NO: 102 EVLLQQSGPEVVKPGASVKISCKASGYKFNDYNIDWVKQSHGKSLDWIGNINPNNGGTIYNQKFKGKATLTVDKSSSTPYMDLRSLTSEDTAVYFCSRERDNRYDRWSAYWGQGTLVTVSAGGGGSGGGGSGGGGGSGGGGSDIVMTQSQKFMSTSVGDRVSVTCKASQNVGANVAWYQHKPG QSPKALIYSASYWNSGVPDRFTGSGSGTDFTLTISSVHSDDLAEYFCQQYNSYPFAFGSGTKLEIKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADS VKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001023-SEQ ID NO: 104 EVQLQQSGPELVKPGASMKISKASGYSFTGYTMNWVKQSHGKNLEWIGLVNVYHGGTTYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCAGTGTGALDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGPSPK RWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWRSNPYTFGGGTKLEIKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSV KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001024-SEQ ID NO: 106 DVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEWVAYISSGSNTIYYADTLKGRFTISRDNPRNTLFLQMTSLRSEDTAIYYCTRRPIPGAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSQIVLTQSPALISAFPGERVTMTCSASSSVTYIHWYQQKPRSSP KSWIYLTSNLASGVPTRFSGSGSGTSYSLTISSMEAEDAATYFCQQWSTNSLTFGAGTKLEIKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSV KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001036-SEQ ID NO: 108 QVQLQQSGAELMKPGASVKIPCKATGYTFSSYWIEWVKQRPGHGLEWIGEVLPGSGSTNYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARPYYRYDVGAWFAYWGQGTLVTVSAGGGGSGGGGSGGGGGSGGGGSDIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSGNQKNYLAWY QQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPYTFGGGTKLEIKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYA DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001038-SEQ ID NO: 110 QVQLQQSGAELAKPGASVKMSCKASGYAFTTYWMHWVKQRPGQGLEWIGYINPTTGYTDYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARWGLPPFAYWGQGTLVTVSAGGGGSGGGGSGGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSITCKASQDVNTAVAWYQQKPGQSP KLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSV KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001040-SEQ ID NO: 112 QVQLKESGPGLVAPSQSLSITCTVSGFSLSRYSIQWVRQPPGKGLEWLGMIWGGGSTDYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARNQGGYDVWFAYWGQGTLVTVSAGGGGSGGGGSGGGSGGGGSDIVMSQSPSSLAVSVGEKVTMNCKSSQSLLYSGNQKKYLAWYQQ KPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAIYYCQQYYSYPFTFGGGTKLEIKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYAD SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001042-SEQ ID NO: 114 QVQLQQSGTELMKPGAAVKISKATGYTISSYWIEWVKQRPGHGLEWIGEILPGSGSINYNEKFKGKATFTADTSSNTAYIQLSSLTSEDSAVYYCARGGIYYGPLYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGGSGGGGSDIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSGNQKNYLAWY QQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPYTFGGGTKLEIKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYA DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001044-SEQ ID NO: 116 EVKLVESGGGLVQPGGALRLSCATSGFTFTDYYMSWVRQPPGKALEWLGFIRNKANGYTTEYSASVKGRFTISRDNSQSILYLQMNTLRAEDSATYYCASHYYGRAWFAYWGQGTLVTVSAGGGGSGGGGSGGGGSGGGGSQIVLTQSPTIMSASPGEKVTMTCSASSSVSNMHWYQQKSGT SPKRWIYDTSTLASGVPARFSGSGSGSYSLTISSMEAEDVATYYCQQWSSNPPTFGGGTTLEIKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADS VKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001027-SEQ ID NO: 118 DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLFSVNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTIGSVKAEDLAVYYCQHYYSYPWTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELMNPGASVRISCKATGYTFSTSWIEWIKQRPGHGLEWIGEI LPGSGSTNYNEKFKGKATFTADTSSNTAYMQLSSLTFEDSAVYYCARGDDGSYVRWYFDVWGAGTTVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYA DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001052-SEQ ID NO: 119 DYKDDDDKDYKDDDDKGGSGGEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGV SDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ >CD4001053-SEQ ID NO: 120 DYKDDDDKDYKDDDDKGGSGGEPPTACSDKQYLHDGQCCDLCQPGSRLTSHCTALEKTQCHPCDSGEFSAQWNREIRCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGV SDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ >CD4001054-SEQ ID NO: 121 DYKDDDDKDYKDDDDKGGSGGEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHRHCEPNQGLRVKKEGTAESDTVCTCKEGQHCTSKDCEACAQHRSCSPGFGVKQIATGV SDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ >CD4001055-SEQ ID NO: 122 DYKDDDDKDYKDDDDKGGSGGEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHTPCIPGFGVMEMATET TDTVCHPCPVGFFSNQSSLFEKCYPWTSCEDKNLEVLQKGTSQTNVICGLKSRMRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ >CD4001056-SEQ ID NO: 123 DYKDDDDKDYKDDDDKGGSGGEPPTACSDKQYLHDGQCCDLCQPGSRLTSHCTALEKTQCHPCDSGEFSAQWNREIRCHQHRHCEPNQGLRVKKEGTAESDTVCTCKEGQHCTSKDCEACAQHTPCIPGFGVMEMATET TDTVCHPCPVGFFSNQSSLFEKCYPWTSCEDKNLEVLQKGTSQTNVICGLKSRMRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ >CD4001238-SEQ ID NO: 124 DYKDDDDKDYKDDDDKGGSGGEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCKEGQHCTSKDCEACAQHRSCSPGFGVKQIATET TDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ >CD4001239-SEQ ID NO: 125 DYKDDDDKDYKDDDDKGGSGGEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHTPCIPGFGVKQIATGV SDTICEPCPVGFFSNQSSLFEKCHPWTSCETKDLVVQQAGTSQTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ >CD4001240-SEQ ID NO: 126 DYKDDDDKDYKDDDDKGGSGGEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVMEMATET TDTVCHPCPVGFFSNVSSAFEKCYPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ >CD4001241-SEQ ID NO: 127 DYKDDDDKDYKDDDDKGGSGGEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGV SDTICEPCPVGFFSNVSSAFEKCYPWTSCEDKNLEVLQKGTSQTNVICGLKSRMRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ >CD4001047-SEQ ID NO: 128 DIVMTQSHKFMSTSVGDRVSITCKASQDVNTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELAKPGASVKMSCKASGYAFTTYWMHWVKQRPGQGLEWIGYINPT TGYTDYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARWGLPPFAYWGQGTLVTVSAEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSV KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001048-SEQ ID NO: 129 DVVMTQTPLSLPVSLGDQASISCRSSQSLVHYNGYTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQTTHVPLTFGAGTKLELRGGGGSGGGGSGGGGSGGGGSEVMLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPERRLEWVASIS SAGTYIYYPDSVKGRFTISRDNAKNILYLQMNSLRSEDTAMYYCARHGNGYDPLWYFDVWGAGTTVTVSSEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYAD SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001049-SEQ ID NO: 130 DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLFSVNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTIGSVKAEDLAVYYCQHYYSYPWTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELMNPGASVRISCKATGYTFSTSWIEWIKQRPGHGLEWIGEI LPGSGSTNYNEKFKGKATFTADTSSNTAYMQLSSLTFEDSAVYYCARGDDGSYVRWYFDVWGAGTTVTVSSEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYA DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001064-SEQ ID NO: 131 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPG KAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIKEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSDIVMTQSHKFMSTSVGDRVSITCKASQDVNTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRF TGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELAKPGASVKMSCKASGYAFTTYWMHWVKQRPGQGLEWIGYINPTTGYTDYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARWGLPPFAYWGQGTLVTVSA >CD4001065-SEQ ID NO: 132 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAP KLLIHYTSSLQPGVPSRFSGSGSGTDFLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIKEPKSSDKTHTCPPCPAAAPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHYNGYTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGS GSGTDFTLKISRVEAEDLGVYFCSQTTHVPLTFGAGTKLELRGGGGSGGGGSGGGGSGGGGSEVMLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPERRLEWVASISSAGTYIYYPDSVKGRFTISRDNAKNILYLQMNSLRSEDTAMYYCARHGNGYDPLWYFDVWGAGTTVTVSS >CD4001066-SEQ ID NO: 133 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAP KLLIHYTSSLQPGVPSRFSGSGSGTDFLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIKEPKSSDKTHTCPPCPAAAPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSDIVMSQSPSSLAVSVGEKVTMSCKSSQSLLFSVNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTG SGSGTDFTLTIGSVKAEDLAVYYCQHYYSYPWTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELMNPGASVRISCKATGYTFSTSWIEWIKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTADTSSNTAYMQLSSLTFEDSAVYYCARGDDGSYVRWYFDVWGAGTTVTVSS >CD4001067_Strand1-SEQ ID NO:134 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVS SGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFG QGTKLEIKEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >CD4001067_Strand2-SEQ ID NO:135 DIVMTQSHKFMSTSVGDRVSITCKASQDVNTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIKGGGGGSGGGGSG GGGSGGGGSQVQLQQSGAELAKPGASVKMSCKASGYAFTTYWMHWVKQRPGQGLEWIGYINPTTGYTDYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARWGLPPFAYWGQG TLVTVSAEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPG >CD4001068_Strand 1-SEQ ID NO: 136 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVS SGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFG QGTKLEIKEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >CD4001068_Strand2-SEQ ID NO:137 DVVMTQTPLSLPVSLGDQASISCRSSQSLVHYNGYTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQTTHVPLTFGAGTKLELRGGGGGSGGGG SGGGGSGGGGSEVMLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPERRLEWVASISSAGTYIYYPDSVKGRFTISRDNAKNILYLQMNSLRSEDTAMYYCARHGNGYDPLWYFD VWGAGTTVTVSSEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPG >CD4001069_Strand 1-SEQ ID NO: 138 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVS SGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFG QGTKLEIKEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >CD4001069_Strand2-SEQ ID NO:139 DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLFSVNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTIGSVKAEDLAVYYCQHYYSYPWTFGGGGTKLEIKGGGGSGGG GSGGGGSGGGGSQVQLQQSGAELMNPGASVRISCKATGYTFSTSWIEWIKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTADTSSNTAYMQLSSLTFEDSAVYYCARGDDGSYVRWYF DVWGAGTTVTVSSEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPG >CD4001070_Strand 1-SEQ ID NO: 140 EPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLV ESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGS GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001070_Strand2-SEQ ID NO:141 DIVMTQSHKFMSTSVGDRVSITCKASQDVNTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELAKPGASVKMSCKASGYAFTTYWMHWVKQRPGQGLEWIGYINPT TGYTDYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARWGLPPFAYWGQGTLVTVSAEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQGNVFSCSVMHEALHNRFTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSV KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001071_Strand 1-SEQ ID NO: 142 EPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLV ESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGS GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001071_Strand2-SEQ ID NO:143 DVVMTQTPLSLPVSLGDQASISCRSSQSLVHYNGYTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQTTHVPLTFGAGTKLELRGGGGSGGGGSGGGGSGGGGSEVMLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPERRLEWVASIS SAGTYIYYPDSVKGRFTISRDNAKNILYLQMNSLRSEDTAMYYCARHGNGYDPLWYFDVWGAGTTVTVSSEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYAD SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001072_Strand 1-SEQ ID NO: 144 EPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLV ESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGS GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001072_Strand2-SEQ ID NO:145 DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLFSVNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTIGSVKAEDLAVYYCQHYYSYPWTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELMNPGASVRISCKATGYTFSTSWIEWIKQRPGHGLEWIGEI LPGSGSTNYNEKFKGKATFTADTSSNTAYMQLSSLTFEDSAVYYCARGDDGSYVRWYFDVWGAGTTVTVSSEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYA DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001073_Strand1-SEQ ID NO:146 DIVMTQSHKFMSTSVGDRVSITCKASQDVNTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIKGGGGGSGGGGSG GGGSGGGGSQVQLQQSGAELAKPGASVKMSCKASGYAFTTYWMHWVKQRPGQGLEWIGYINPTTGYTDYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARWGLPPFAYWGQG TLVTVSAEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >CD4001073_Strand2-SEQ ID NO:147 DIVMTQSHKFMSTSVGDRVSITCKASQDVNTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELAKPGASVKMSCKASGYAFTTYWMHWVKQRPGQGLEWIGYINPT TGYTDYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARWGLPPFAYWGQGTLVTVSAEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQGNVFSCSVMHEALHNRFTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSV KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001074_Strand 1-SEQ ID NO: 148 DVVMTQTPLSLPVSLGDQASISCRSSQSLVHYNGYTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQTTHVPLTFGAGTKLELRGGGGGSGGGG SGGGGSGGGGSEVMLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPERRLEWVASISSAGTYIYYPDSVKGRFTISRDNAKNILYLQMNSLRSEDTAMYYCARHGNGYDPLWYFD VWGAGTTVTVSSEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >CD4001074_Strand2-SEQ ID NO:149 DVVMTQTPLSLPVSLGDQASISCRSSQSLVHYNGYTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQTTHVPLTFGAGTKLELRGGGGSGGGGSGGGGSGGGGSEVMLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPERRLEWVASIS SAGTYIYYPDSVKGRFTISRDNAKNILYLQMNSLRSEDTAMYYCARHGNGYDPLWYFDVWGAGTTVTVSSEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYAD SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001075_Strand1-SEQ ID NO:150 DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLFSVNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTIGSVKAEDLAVYYCQHYYSYPWTFGGGGTKLEIKGGGGSGGG GSGGGGSGGGGSQVQLQQSGAELMNPGASVRISCKATGYTFSTSWIEWIKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTADTSSNTAYMQLSSLTFEDSAVYYCARGDDGSYVRWYF DVWGAGTTVTVSSEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >CD4001075_Strand2-SEQ ID NO:151 DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLFSVNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTIGSVKAEDLAVYYCQHYYSYPWTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELMNPGASVRISCKATGYTFSTSWIEWIKQRPGHGLEWIGEI LPGSGSTNYNEKFKGKATFTADTSSNTAYMQLSSLTFEDSAVYYCARGDDGSYVRWYFDVWGAGTTVTVSSEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGGGGSPSEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYA DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001076_Strand1-SEQ ID NO:152 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVS SGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFG QGTKLEIKEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >CD4001076_Strand2-SEQ ID NO:153 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPG KAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIKEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGGGGSPSDIVMTQSHKFMSTSVGDRVSITCKASQDVNTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRF TGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELAKPGASVKMSCKASGYAFTTYWMHWVKQRPGQGLEWIGYINPTTGYTDYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARWGLPPFAYWGQGTLVTVSA >CD4001077_Strand1-SEQ ID NO:154 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVS SGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFG QGTKLEIKEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >CD4001077_Strand2-SEQ ID NO:155 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAP KLLIHYTSSLQPGVPSRFSGSGSGTDFLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIKEPKSSDKTHTCPPCPAAAPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGGGGSPSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHYNGYTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGS GSGTDFTLKISRVEAEDLGVYFCSQTTHVPLTFGAGTKLELRGGGGSGGGGSGGGGSGGGGSEVMLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPERRLEWVASISSAGTYIYYPDSVKGRFTISRDNAKNILYLQMNSLRSEDTAMYYCARHGNGYDPLWYFDVWGAGTTVTVSS >CD4001078_Strand1-SEQ ID NO:156 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVS SGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFG QGTKLEIKEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >CD4001078_Strand2-SEQ ID NO:157 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAP KLLIHYTSSLQPGVPSRFSGSGSGTDFLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIKEPKSSDKTHTCPPCPAAAPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGGGGSPSDIVMSQSPSSLAVSVGEKVTMSCKSSQSLLFSVNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTG SGSGTDFTLTIGSVKAEDLAVYYCQHYYSYPWTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELMNPGASVRISCKATGYTFSTSWIEWIKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTADTSSNTAYMQLSSLTFEDSAVYYCARGDDGSYVRWYFDVWGAGTTVTVSS >CD4001079_Strand 1-SEQ ID NO: 158 EPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSDIVM TQSHKFMSTSVGDRVSITCKASQDVNTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIKGGGGSGGGGSGGGSGG GGSQVQLQQSGAELAKPGASVKMSCKASGYAFTTYWMHWVKQRPGQGLEWIGYINPTTGYTDYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARWGLPPFAYWGQGTLVTVSA >CD4001079_Strand2-SEQ ID NO:159 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPG KAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIKEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGGGGSPSDIVMTQSHKFMSTSVGDRVSITCKASQDVNTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRF TGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELAKPGASVKMSCKASGYAFTTYWMHWVKQRPGQGLEWIGYINPTTGYTDYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARWGLPPFAYWGQGTLVTVSA >CD4001080_Strand 1-SEQ ID NO: 160 EPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSDVVMTQTPL SLPVSLGDQASISCRSSQSLVHYNGYTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQTTHVPLTFGAGTKLELRGGGGSGGGGSGGGGGSGGGG SEVMLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPERRLEWVASISSAGTYIYYPDSVKGRFTISRDNAKNILYLQMNSLRSEDTAMYYCARHGNGYDPLWYFDVWGAGTTVTVSS >CD4001080_Strand2-SEQ ID NO:161 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAP KLLIHYTSSLQPGVPSRFSGSGSGTDFLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIKEPKSSDKTHTCPPCPAAAPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGGGGSPSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHYNGYTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGS GSGTDFTLKISRVEAEDLGVYFCSQTTHVPLTFGAGTKLELRGGGGSGGGGSGGGGSGGGGSEVMLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPERRLEWVASISSAGTYIYYPDSVKGRFTISRDNAKNILYLQMNSLRSEDTAMYYCARHGNGYDPLWYFDVWGAGTTVTVSS >CD4001081_Strand 1-SEQ ID NO: 162 EPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSDIVMSQSPS SLAVSVGEKVTMSCKSSQSLLFSVNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTIGSVKAEDLAVYYCQHYYSYPWTFGGGTKLEIKGGGGSGGGGSGGGGGSGGGG SQVQLQQSGAELMNPGASVRISCKATGYTFSTSWIEWIKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTADTSSNTAYMQLSSLTFEDSAVYYCARGDDGSYVRWYFDVWGAGTTVTVSS >CD4001081_Strand2-SEQ ID NO:163 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAP KLLIHYTSSLQPGVPSRFSGSGSGTDFLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIKEPKSSDKTHTCPPCPAAAPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGGGGSPSDIVMSQSPSSLAVSVGEKVTMSCKSSQSLLFSVNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTG SGSGTDFTLTIGSVKAEDLAVYYCQHYYSYPWTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELMNPGASVRISCKATGYTFSTSWIEWIKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTADTSSNTAYMQLSSLTFEDSAVYYCARGDDGSYVRWYFDVWGAGTTVTVSS >POC01043-SEQ ID NO: 164 EVQLQESGPGLVKPSETLSLTTCTVSGYSITSNYYWNWIRQPPGKGLEWMGYIRYDGSNNYNPSLKNRVTISRDTSKNQFSLKLSSVTAADTAVYYCARLDYWGQGTTVTVSSDLSGGGGSGGGGSGGGGSGGGGSTGDAVMTQTPLSLSVTPGQPASISCRSSQSLENTNGNTFLNWYLQK PGQSPQLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYYCLQVTHVPFTFGQGTKLEIKEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS >PDL01029-SEQ ID NO: 165 DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKGGGGGSGGGGSG GGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLV TVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >PDL01085-SEQ ID NO: 166 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSS GGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQG TKLEIKSSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >PDL01127_light chain-SEQ ID NO: 167 EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >PDL01127_heavy chain-SEQ ID NO: 168 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGG GGSGGGGSNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQS IHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLGGGGSGGGSQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASS QAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLGGGGSGGGSQIAAHVISEASSKTTSVLQWAEKGYYTM SNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL >PDL01153-SEQ ID NO: 169 SSEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQL VESGGGLVKPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVAFITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSGGGGS GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIK >CD4001086-SEQ ID NO: 170 EVVMTQSPGTLSLSPGERATLSCRSSQSLVHYNGYTYLHWYQQKPGQAPRLLIYKVSNRFSGIPARFSGSGSGTDFTLTISRLEPEDLAVYFCSQTTHVPLTFGQGTKVEIKGGGGSGGGGSGGGS GGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASISSAGTYIYYADSVQGRFTISRDNGKNSLYLQMNSLRAEDTALYYCARHGNGYDPLWYFDVWGQGTMVTVSS >CD4001087-SEQ ID NO: 171 EVVMTQSPDTLSVSPGERATLSCRSSQSLVHYNGYTYLHWYQQKPGQVPRLLIYKVSNRFSGVPARFTGSGSGTEFTLTISSLQSEDFAVYFCSQTTHVPLTFGQGTKLEIKGGGGSGGGGSGGGS GGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASISSAGTYIYYADSVQGRFTISRDNGKNSLYLQMNSLRAEDTALYYCARHGNGYDPLWYFDVWGQGTMVTVSS >CD4001088-SEQ ID NO: 172 EVVMTQSPDFQSVTPKEKVTITCRSSQSLVHYNGYTYLHWYQQKPDQSPKLLIYKVSNRFSGVPSRFSGSGSGTDFTLTINSLEAEDAAAYFCSQTTHVPLTFGPGTKVDIKGGGGSGGGGSGGGGS GGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASISSAGTYIYYADSVQGRFTISRDNGKNSLYLQMNSLRAEDTALYYCARHGNGYDPLWYFDVWGQGTMVTVSS >CD4001089-SEQ ID NO: 173 DVQMTQSPILLSASVGDRVTITCRSSQSLVHYNGYTYVHWYQQRTNGSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISSLQPEDIADYFCQQTTHVPLTFGAGTKVEIKGGGGSGGGGSGGGS GGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASISSAGTYIYYADSVQGRFTISRDNGKNSLYLQMNSLRAEDTALYYCARHGNGYDPLWYFDVWGQGTMVTVSS >CD4001090-SEQ ID NO: 174 EVVMTQSPDFQSVTPKEKVTITCRSSQSLVHYNGYTYLHWYQQKPDQSPKLLIYKVSNRFSGVPSRFSGSGSGTDFTLTINSLEAEDAAAYFCSQTTHVPLTFGPGTKVDIKGGGGSGGGGSGGGGS GGGGSEVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASISSAGTYIYYADSVKGRFTISRDNSKNTLYLQMNGLRAEDTAVYYCARHGNGYDPLWYFDVWGQGTLVTVSS >CD4001091-SEQ ID NO: 175 DVQMTQSPILLSASVGDRVTITCRSSQSLVHYNGYTYVHWYQQRTNGSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISSLQPEDIADYFCQQTTHVPLTFGAGTKVEIKGGGGSGGGGSGGGS GGGGSEVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASISSAGTYIYYADSVKGRFTISRDNSKNTLYLQMNGLRAEDTAVYYCARHGNGYDPLWYFDVWGQGTLVTVSS >CD4001092-SEQ ID NO: 176 DVVMTQSPDSLAVSLGERATINCRSSQSLVHYNGYTYLHWYQQKPGQPPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCSQTTHVPLTFGAGTKLELRGGGGSGGGGSGGGS GGGGSEVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASISSAGTYIYYADSVKGRFTISRDNSKNTLYLQMNGLRAEDTAVYYCARHGNGYDPLWYFDVWGQGTLVTVSS >CD4001093-SEQ ID NO: 177 DVVMTQSPDTLSLSPGERATLSCRSSQSLVHYNGYTYLHWYQQQPGQAPRLLIYKVSNRFSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYFCSQTTHVPLTFGQGTKLEIKGGGGSGGGGSGGGS GGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASISSAGTYIYYTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCARHGNGYDPLWYFDVWGQGTTVTVSS >CD4001094-SEQ ID NO: 178 DVQMTQSPILLSASVGDRVTITCRSSQSLVHYNGYTYVHWYQQRTNGSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISSLQPEDIADYFCQQTTHVPLTFGAGTKVEIKGGGGSGGGGSGGGS GGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASISSAGTYIYYTDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCARHGNGYDPLWYFDVWGQGTTVTVSS >CD4001095-SEQ ID NO: 179 DVVMTQSPDTLSLSPGERATLSCRSSQSLVHYNGYTYLHWYQQQPGQAPRLLIYKVSNRFSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYFCSQTTHVPLTFGQGTKLEIKGGGGSGGGGSGGGS GGGGSQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLVWVASISSAGTYIYYADSVKGRFTISRDNAKNTLYLQMTSLRAEDTAIYYCARHGNGYDPLWYFDVWGQGALVTVSS >CD4001096-SEQ ID NO: 180 DVQMTQSPILLSASVGDRVTITCRSSQSLVHYNGYTYVHWYQQRTNGSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISSLQPEDIADYFCQQTTHVPLTFGAGTKVEIKGGGGSGGGGSGGGS GGGGSQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLVWVASISSAGTYIYYADSVKGRFTISRDNAKNTLYLQMTSLRAEDTAIYYCARHGNGYDPLWYFDVWGQGALVTVSS >CD4001097-SEQ ID NO: 181 EVVMTQSPGTLSLSPGERATLSCRSSQSLVHYNGYTYLHWYQQKPGQAPRLLIYKVSNRFSGIPARFSGSGSGTDFTLTISRLEPEDLAVYFCSQTTHVPLTFGQGTKVEIKGGGGSGGGGSGGGS GGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASISSAGTYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHGNGYDPLWYFDVWGAGTTVTVSS >CD4001098-SEQ ID NO: 182 DVQMTQSPILLSASVGDRVTITCRSSQSLVHYNGYTYVHWYQQRTNGSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISSLQPEDIADYFCQQTTHVPLTFGAGTKVEIKGGGGSGGGGSGGGS GGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASISSAGTYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHGNGYDPLWYFDVWGAGTTVTVSS >CD4001099-SEQ ID NO: 183 EVVMTQSPDFQSVTPKEKVTITCRSSQSLVHYNGYTYLHWYQQKPDQSPKLLIYKVSNRFSGVPSRFSGSGSGTDFTLTINSLEAEDAAAYFCSQTTHVPLTFGPGTKVDIKGGGGSGGGGSGGGGS GGGGSEVQLVESGGGLEQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASISSAGTYIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGNGYDPLWYFDVWGQGTTVTVSS >CD4001100-SEQ ID NO: 184 DVQMTQSPILLSASVGDRVTITCRSSQSLVHYNGYTYVHWYQQRTNGSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISSLQPEDIADYFCQQTTHVPLTFGAGTKVEIKGGGGSGGGGSGGGS GGGGSEVQLVESGGGLEQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASISSAGTYIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGNGYDPLWYFDVWGQGTTVTVSS >CD4001132-SEQ ID NO: 185 DVQMTQSPILLSASVGDRVTITCRSSQSLVHYNGYTYVHWYQQKPGKAPKLLIYKVSNLFSGVPSRFSGSGSGTDFTLTISSLQPEDIADYFCQQTTHVPLTFGAGTKVEIKGGGGSGGGGSGGGS GGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISSAGTYIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGNGYDPLWYFDVWGQGTTVTVSS >CD4001133_humanized5B9_VH-SEQ ID NO: 186 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISSAGTYIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGNGYDPLWYFDVWGQGTTVTVSS >Humanized 5B9_HCDR1-SEQ ID NO: 187-SYAMS Humanized 5B9_HCDR2 - SEQ ID NO: 188 - AISSAGTYIYYADSVKG Humanized 5B9_HCDR3-SEQ ID NO: 189-HGNGYDPLWYFDV >CD4001133_humanized5B9_VL-SEQ ID NO: 190 DIQMTQSPSSLSASVGDRVTITCRASQSLVHYNGYTYLNWYQQKPGKAPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQTTHVPLTFGQGTKVEIK >Humanized 5B9_LCDR1-SEQ ID NO: 191-RASQSLVHYNGYTYLN >Humanized 5B9_LCDR2-SEQ ID NO: 192-KVSNRFS Humanized 5B9_LCDR3-SEQ ID NO: 193-QQTTHVPLT >CD4001209-SEQ ID NO: 194 SSEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSDIQMTQSP SSLSASVGDRVTITCRASQSLVHYNGYTYLNWYQQKPGKAPKLLIYKVSNRFSGVPDRFSGSGSGTFTLTISSLQPEDFATYYCQQTTHVPLTFGQGTKVEIKGGGGSGGGGSGGGSGGGG SEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISSAGTYIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGNGYDPLWYFDVWGQGTTVTVSSS >PDL01157_Strand 1-SEQ ID NO: 195 DIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQSGVPSRFSGSGSGTFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVSFITSLAYI IYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSSEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSQVQLVQSGAEVKKPGASVKVSCKASGYTFTRSTMHWVRQAPGQGLEWIGYINPSSAYTNYAQKFQG RVTLTADKSTSTAYMELSSLRSEDTAVYYCASPQVHYDYNGFPYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASSSVSYMNWYQQKPGKAPKRWIYDSSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSRNPPTFGQGTKVEIKRS >PDL01157_Strand2-SEQ ID NO:196 DIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIKGGGGGSGGGGSGGG GSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVSFITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQ GTLVTVSSSEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPG >PDL01158_Strand 1-SEQ ID NO: 197 DIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQSGVPSRFSGSGSGTFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVSFITSLAYI IYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSSEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQGNVFSCSVMHEALHNHYTQKSLSPGGGGSPSQVQLVQSGPEVKKPGSSVKVSCKASGYTFSRSTMHWVRQAPGQGLEWIGYINPSSAYTNYNQKFKD RVTITADKSTSTAYMELSSLRSEDTAVYYCARPQVHYDYNGFPYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTMTCSASSSVSYMNWYQQKPGKAPKRWIYDSSKLASGVPSRFSGSGSGTDYTLTISSLQPDDFATYYCQQWSRNPPTFGGGTKVEIKRS >PDL01158_Strand2-SEQ ID NO:198 DIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQSGVPSRFSGSGSGTFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVSFITSLAYI IYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQGTLVTVSSSEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSQVQLVQSGAEVKKPGASVKVSCKASGYTFTRSTMHWVRQAPGQGLEWIGYINPSSAYTNYAQKFQG RVTLTADKSTSTAYMELSSLRSEDTAVYYCASPQVHYDYNGFPYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASSSVSYMNWYQQKPGKAPKRWIYDSSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSRNPPTFGQGTKVEIKRS >PC401020-SEQ ID NO: 199 DIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQSGVPSRFSGSGSGTFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVSFITSLAY I ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQGNVFSCSVMHEALHNHYTQKSLSPGGGGSPSQVQLVQSGPEVKKPGSSVKVSCKASGYTFSRSTMHWVRQAPGQGLEWIGYINPSSAYTNYNQKFKD RVTITADKSTSTAYMELSSLRSEDTAVYYCARPQVHYDYNGFPYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTMTCSASSSVSYMNWYQQKPGKAPKRWIYDSSKLASGVPSRFSGSGSGTDYTLTISSLQPDDFATYYCQQWSRNPPTFGGGTKVEIKRS >PC401021-SEQ ID NO: 200 DIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQSGVPSRFSGSGSGTFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVSFITSLAY I ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSEVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGNIYPSGGSTNYAQKFQ GRVTMTVDTSTSTVYMELSSLRSEDTAVYYCASFSDGYYAYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGGSGGGGSEIVMTQSPATLSLSPGERATLSCRASQSVSSYLNWYQQKPGQAPRLLIYYASRRHTGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCQQGYNLPYTFGQGTKVEIK >PC401026-SEQ ID NO: 201 EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGNIYPSGGSTNYAQKFQGRVTMTVDTSTSTVYMELSSLRSEDTAVYYCASFSDGYYAYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGGSGGGGSEIVMTQSPATLSLSPGERATLSCRASQSVSSYLNWYQQKPGQA PRLLIYYASRRHTGIPARFSGSGTDFTLTISSLQPEDFAVYYCQQGYNLPYTFGQGTKVEIKEPKSSDKTHTCPPCPAAAPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSDIQMTQSPSSVSASVGDRVTITCRASQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASSLQSGVPSRFSG SGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWIEWIRQAPGQGLEWMGSILPGSGSTNYAQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCARGDAGSYVRWYFDHWGQGTLVTVSS >CD4001101-SEQ ID NO: 202 DIQMTQSPSTLSASVGDRVTITCKSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPARFSGSGSGTEFTLTISSLQPDDFATYYCQHYYSYPWTFGQGTKVEVKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSTSWIEWIKQAPGQGLEWIGEILPGSGSTNYNENFRNKATFTADTSISTAYMELSRLRSDDTAVYYCARGDDGSYVRWYFDVWGQGTLVTVSS >CD4001102-SEQ ID NO: 203 DIQMTQSPSSLSASVGDRVTITCKSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQHYYSYPWTFGGGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSTSWIEWIKQAPGQGLEWIGEILPGSGSTNYNENFRNKATFTADTSISTAYMELSRLRSDDTAVYYCARGDDGSYVRWYFDVWGQGTLVTVSS >CD4001103-SEQ ID NO: 204 QIVMTQSPGTLSLSPGERATMTCKSSQSLLFSVNQKNYLAWYQQKPGQAPRLLIYWASTRESGVPDRFSGSGSGTDFTLTISRLEPEDAATYYCQHYYSYPWTFGAGTKLEIKGGGGSGGGGSGGGG SGGGGSQVKLVQSGAEVKKPGASVKVSCKASGYTFSTSWIEWIRQAPGQGLEWIGEILPGSGSTNYAQKFQGRATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDVWGQGTTVTVSS >CD4001134-SEQ ID NO: 205 DIVMTQSPDSLALSLGERATMNCKSSQSLLFSVNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHYYSYPWTFGQGTKLEIKGGGGSGGGGSGGGG SGGGGSQVKLVQSGAEVKKPGASVKVSCKASGYTFSTSWIEWIRQAPGQGLEWIGEILPGSGSTNYAQKFQGRATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDVWGQGTTVTVSS >CD4001135-SEQ ID NO: 206 DIVMTQSPDSLALSLGERATMNCKSSQSLLFSVNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHYYSYPWTFGQGTKLEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSTSWIEWVRQAPGQGLEWIGEILPGSGSTNYAQKFQGRATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDVWGQGTTVTVSS >CD4001136-SEQ ID NO: 207 DIVMTQSPDSLALSLGERATMNCKSSQSLLFSVNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHYYSYPWTFGQGTKLEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSTSWMEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDDGSYVRWYFDVWGQGTTVTVSS >CD4001137-SEQ ID NO: 208 QIVMTQSPGTLSLSPGERATMTCKSSQSLLFSVNQKNYLAWYQQKPGQAPRLLIYWASTRESGVPDRFSGSGSGTDFTLTISRLEPEDAATYYCQHYYSYPWTFGAGTKLEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSTSWMEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDDGSYVRWYFDVWGQGTTVTVSS >CD4001138-SEQ ID NO: 209 DIVMTQSPDSLAVSLGERATINCKSSQSLLFSVNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSTSWMEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDDGSYVRWYFDVWGQGTTVTVSS >CD4001152-SEQ ID NO: 210 QIVMTQSPDSLALSLGERATMNCKSSQSLLFSVNQKNYLAWYQQKPGQAPRLLIYWASTRESGVPDRFSGSGSGTDFTLTISRLEPEDVATYYCQHYYSYPWTFGAGTKLEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSTSWIEWVRQAPGQGLEWIGEILPGSGSTNYAQKFQGRATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDVWGQGTTVTVSS >CD4001153-SEQ ID NO: 211 DIVMTQSPDSLALSLGERATMNCKSSQSLLFSVNQKNYLAWYQQKPGQAPRLLIYWASTRESGVPDRFSGSGSGTDFTLTISRLEPEDVATYYCQHYYSYPWTFGAGTKLEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSTSWIEWVRQAPGQGLEWIGEILPGSGSTNYAQKFQGRATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDVWGQGTTVTVSS >CD4001154-SEQ ID NO: 212 QIVMTQSPDSLALSLGERATMNCKSSQSLLFSVNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISRLEPEDVATYYCQHYYSYPWTFGAGTKLEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSTSWIEWVRQAPGQGLEWIGEILPGSGSTNYAQKFQGRATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDVWGQGTTVTVSS >CD4001155-SEQ ID NO: 213 QIVMTQSPDSLALSLGERATMNCKSSQSLLFSVNQKNYLAWYQQKPGQAPRLLIYWASTRESGVPDRFSGSGSGTDFTLTISRLEPEDVAVYYCQHYYSYPWTFGQGTKLEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSTSWIEWVRQAPGQGLEWIGEILPGSGSTNYAQKFQGRATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDVWGQGTTVTVSS >CD4001156-SEQ ID NO: 214 QIVMTQSPDSLALSLGERATMNCKSSQSLLFSVNQKNYLAWYQQKPGQAPRLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVATYYCQHYYSYPWTFGAGTKLEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSTSWIEWVRQAPGQGLEWIGEILPGSGSTNYAQKFQGRATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDVWGQGTTVTVSS >CD4001157-SEQ ID NO: 215 QIVMTQSPDSLAVSLGERATINCKSSQSLLFSVNQKNYLAWYQQKPGQAPRLLIYWASTRESGVPDRFSGSGSGTDFTLTISRLEPEDVATYYCQHYYSYPWTFGAGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSTSWIEWVRQAPGQGLEWIGEILPGSGSTNYAQKFQGRATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDVWGQGTTVTVSS >CD4001158-SEQ ID NO: 216 QIVMTQSPDSLALSLGERATMNCKSSQSLLFSVNQKNYLAWYQQKPGQAPRLLIYWASTRESGVPDRFSGSGSGTDFTLTISRLEPEDVATYYCQHYYSYPWTFGAGTKLEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSTSWMEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDDGSYVRWYFDVWGQGTTVTVSS >CD4001160-SEQ ID NO: 217 QIVMTQSPDSLALSLGERATMNCKSSQSLLFSVNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISRLEPEDVATYYCQHYYSYPWTFGAGTKLEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSTSWMEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDDGSYVRWYFDVWGQGTTVTVSS >CD4001161-SEQ ID NO: 218 QIVMTQSPDSLALSLGERATMNCKSSQSLLFSVNQKNYLAWYQQKPGQAPRLLIYWASTRESGVPDRFSGSGSGTDFTLTISRLEPEDVAVYYCQHYYSYPWTFGQGTKLEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSTSWMEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDDGSYVRWYFDVWGQGTTVTVSS >CD4001162-SEQ ID NO: 219 QIVMTQSPDSLALSLGERATMNCKSSQSLLFSVNQKNYLAWYQQKPGQAPRLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVATYYCQHYYSYPWTFGAGTKLEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSTSWMEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDDGSYVRWYFDVWGQGTTVTVSS >CD4001163-SEQ ID NO: 220 QIVMTQSPDSLAVSLGERATINCKSSQSLLFSVNQKNYLAWYQQKPGQAPRLLIYWASTRESGVPDRFSGSGSGTDFTLTISRLEPEDVATYYCQHYYSYPWTFGAGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSTSWMEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDDGSYVRWYFDVWGQGTTVTVSS >CD4001164-SEQ ID NO: 221 QIVMTQSPGTLSLSPGERATMTCKSSQSLLFSVNQKNYLAWYQQKPGQAPRLLIYWASTRESGVPDRFSGSGSGTDFTLTISRLEPEDAATYYCQHYYSYPWTFGAGTKLEIKGGGGSGGGGSGGGG SGGGGSQVKLVQSGAEVKKPGASVKVSCKASGYTFSTSWIEWIRQAPGQGLEWIGEILPGSGSTNYAQKFQGRATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDVWGQGTTVTVSS >CD4001171-SEQ ID NO: 222 DIQMTQSPSTLSASVGDRVTITCKSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQHYYSYPWTFGQGTKVEVKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAELKKPGSSVKVSCKASGYTFSTSWIEWIKQAPGQGLEWIGRILPGSGSVHYNQDFKDKATFTADTSTNTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDVWGQGTLVTVSS >CD4001172-SEQ ID NO: 223 DIVMTQSPDSLAVSLGERATINCKSSQSLLFSVNQKNYLAWYQQKPGQPPKLLIYWASYRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHYYSYPWTFGGGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSTSWIEWIRQAPGQGLEWIGNILPGSGSTNYNEKFKNRATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDVWGQGTLVTVSS >CD4001173-SEQ ID NO: 224 EIVMTQSPATLSLSPGERATLSCRSSQSLLFSVNQKNYIAWYQQKPGQAPRLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLEPEDFAVYYCQHYYSYPWTFGQGTKLEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGASVKVSCKATGYTFSTSWIEWIRQAPGQRLEWIGRILPGSGSTNYNEKFKGKATFTADTSASTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDFWGQGTLVTVSS >CD4001174-SEQ ID NO: 225 DIQMTQSPSSLSASVGDRVTITCQSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTFTISSLQPEDIATYYCQHYYSYPWTFGGGTKVEIKGGGGSGGGGSGGGG SGGGGSQVKLVQSGAEVKKPGASVKVSCKATGYTFSTSWIEWIRQAPGQGLEWIGEILPGSGSTNYNEKFKGKATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDVWGQGTTVTVSS >CD4001175-SEQ ID NO: 226 DIQMTQSPSSLSASVGDRVTITCRSSQSLLFSVNQKNYVAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFEQGTKVEIKGGGGSGGGGSGGGG SGGGGSEVQLVESGGGLVQPGGSLRLSCAATGYTFSTSWIEWIRQAPGKGLEWIGLILPGSGSTNYNEKFKGKATFSANTSKNTAYLQMNSLRAEDTAVYYCARGDDGSYVRWYFDYWGQGTLVTVSS >CD4001176-SEQ ID NO: 227 DIQMTQSPSSLSASVGDRVTITCRSSQSLLFSVNQKNYVAWYQQKPGKAPKLLIYWASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYTFSTSWIEWIRQAPGKGLEWIGRILPGSGSTNYADSVKGRATFSADTSKNTAYLQMNSLRAEDTAVYYCARGDDGSYVRWYFDVWGQGTLVTVSS >CD4001177-SEQ ID NO: 228 EIVMTQSPDFQSVTPKEKVTITCRSSQSLLFSVNQKNYLAWYQQKPDQSPKLLIYWASQSFSGVPSRFSGSGSGTFTLTINSLEAEDAAAYYCQHYYSYPWTFGPGTKVDIKGGGGSGGGGSGGGG SGGGGSQVQLVESGGGVVQPGRSLRLSCAASGYTFSTSWIEWIRQAPAKGLEWIGIILPGSGSTYYADSVKGRATFSADTSKNTAYLQMNGLRAEDTAVYYCARGDDGSYVRWYFDVWGQGTLVTVSS >CD4001178-SEQ ID NO: 229 DIQMTQSPSSLSASVGDRVTITCKSSQSLLFSVNQKNYVAWYQQKPGKAPKLLIYWASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYTFSTSWIEWIRQAPGKGLEWIGDILPGSGSTIYNQRFKGRATFSADTSKNTAYLQMNSLRAEDTAVYYCARGDDGSYVRWYFDVWGQGTLVTVSS >CD4001179-SEQ ID NO: 230 DIVMTQSPDSLAVSLGERATINCKSSQSLLFSVNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHYYSYPWTFGGGTKLEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWIEWIRQAPGQGLEWIGGILPGSGSTNYAQKFQGRATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDVWGAGTTVTVSS >CD4001180-SEQ ID NO: 231 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKATGYTFSTSWIEWIRQAPGQGLEWIGSILPGSGSTNYNEKFKGKATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001181-SEQ ID NO: 232 DIQMTQSPSSVSASVGDRVTITCRASQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKATGYTFSTSWIEWIRQAPGQGLEWIGSILPGSGSTNYNEKFKGKATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001182-SEQ ID NO: 233 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASSRQSGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKATGYTFSTSWIEWIRQAPGQGLEWIGSILPGSGSTNYNEKFKGKATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001183-SEQ ID NO: 234 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKATGYTFSTSWIEWIRQAPGQGLEWIGSILPGSGSTNYNEKFKGKATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001184-SEQ ID NO: 235 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWIEWIRQAPGQGLEWIGSILPGSGSTNYNEKFKGKATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001185-SEQ ID NO: 236 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWISWVRQAPGQGLEWIGGILPGSGSTNYNEKFKGKATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001186-SEQ ID NO: 237 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWIEWIRQAPGQGLEWMGSILPGSGSTNYNEKFKGKVTITADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001187-SEQ ID NO: 238 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWIEWIRQAPGQGLEWIGSILPGSGSTNYAEKFKGKATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001188-SEQ ID NO: 239 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWIEWIRQAPGQGLEWIGSILPGSGSTNYNQKFQGRATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001189-SEQ ID NO: 240 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWIEWIRQAPGQGLEWIGSILPGSGSTNYNEKFKGKATFTADESTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001190-SEQ ID NO: 241 DIQMTQSPSSVSASVGDRVTITCRASQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASSRQSGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKATGYTFSTSWIEWIRQAPGQGLEWIGSILPGSGSTNYNEKFKGKATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001191-SEQ ID NO: 242 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKATGYTFSTSWIEWIRQAPGQGLEWIGSILPGSGSTNYNEKFKGKATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001192-SEQ ID NO: 243 DIQMTQSPSSVSASVGDRVTITCRASQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKATGYTFSTSWIEWIRQAPGQGLEWIGSILPGSGSTNYNEKFKGKATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001193-SEQ ID NO: 244 DIQMTQSPSSVSASVGDRVTITCRASQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKATGYTFSTSWIEWIRQAPGQGLEWIGSILPGSGSTNYNEKFKGKATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001194-SEQ ID NO: 245 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWISWVRQAPGQGLEWMGGILPGSGSTNYNEKFKGKVTITADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001195-SEQ ID NO: 246 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWISWVRQAPGQGLEWIGGILPGSGSTNYAEKFKGKATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001196-SEQ ID NO: 247 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWISWVRQAPGQGLEWIGGILPGSGSTNYNQKFQGRATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001197-SEQ ID NO: 248 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWISWVRQAPGQGLEWIGGILPGSGSTNYNEKFKGKATFTADESTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001198-SEQ ID NO: 249 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWIEWIRQAPGQGLEWMGSILPGSGSTNYAEKFKGKVTITADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001199-SEQ ID NO: 250 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWIEWIRQAPGQGLEWMGSILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001200-SEQ ID NO: 251 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWIEWIRQAPGQGLEWIGSILPGSGSTNYAQKFQGRATFTADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001201-SEQ ID NO: 252 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWIEWIRQAPGQGLEWIGSILPGSGSTNYAEKFKGKATFTADESTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001202-SEQ ID NO: 253 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWIEWIRQAPGQGLEWIGSILPGSGSTNYNQKFQGRATFTADESTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001203-SEQ ID NO: 254 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWISWVRQAPGQGLEWMGGILPGSGSTNYAEKFKGKVTITADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001204-SEQ ID NO: 255 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWISWVRQAPGQGLEWMGGILPGSGSTNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001205-SEQ ID NO: 256 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWISWVRQAPGQGLEWMGGILPGSGSTNYNEKFKGKVTITADESTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001206-SEQ ID NO: 257 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWIEWIRQAPGQGLEWMGSILPGSGSTNYAQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001207-SEQ ID NO: 258 DIQMTQSPSSVSASVGDRVTITCRSSQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWIEWIRQAPGQGLEWMGSILPGSGSTNYAEKFKGKVTITADESTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS Humanized 1D5_VH_DNA - SEQ ID NO: 259 CAGGTTCAACTGGTGCAAAGCGGGGCTGAGGTAAAAAAGCCCGGTAGCTCTGTGAAGGTGTCTTGTAAAGCCAGCGGATATACCTTTTCAACTTCCTGGATTGAGTGGATAAGGCAAGCCCCAGGGCAGGGGCTGGAGTGGATGGGCTCTATTCTGCCTGGGAGCGGATCAACTAATTATGCCCAGAAATTTCAAGGTCGGGTTACTATAACAGCAGACACCTCTACAAGTACCGCCTACATGGAGCTGAGTTCCTTGCGATCTGAGGATACAGCCGTATATTATTGTGCAAGGGGTGACGACGGAAGCTACGTCCGATGGTACTTCGATCATTGGGGACAGGGAACTCTCGTAACCGTATCATCT >Humanized 1D5_VL_DNA - SEQ ID NO: 260 GATATACAGATGACCCAATCCCCTAGTTCCGTCTCAGCTTCAGTTGGCGACAGAGTAACTATTACATGCCGTGCTAGCCAATCCCTTCTTTTCTCAGTGAATCAGAAAAACTATCTGGCATGGTATCAACAAAAGCCCGGCAAGGCCCCCAAGCTCCTCATTTACTGGGCTAGCTCATTGCAGTCAGGAGTTCCAAGCCGTTTTTCCGGGTCAGGGTCTGGCACCGATTTTACTTTGACCATCAGTTCTTTGCAACCCGAAGACTTTGCTACATATTACTGTCAACATTACTATAGTTATCCCTGGACATTTGGACAAGGAACCAAAGTCGAGATTAAA >PDL01041_2C11LH - SEQ ID NO: 261 DIQMTQSPASLSASVGETTVTITCRASENIYSFLAWYQQKQGKSPQLLVYKAKTLTEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGSPPETFGGGTKVEIKGGGGSGGGGSGGGGSG GGGSDVKLVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGNYTYYPDSVKGRFTISRDNAKNTLYLQLSSLKSEDTAMYYCTRDQAYYGNLFTYWGQGTLVTVSA >PDL01042_3G5LH-SEQ ID NO: 262 DIQMTQTTSSLSASLGDRVTISCSASQGIRNYLNWYQQKPDGTVKLLIYYTSTLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPWTFGGGTRLEIKGGGGSGGGGSGGGG SGGGGSQIQLQQSGPELVKPGASVKISCKASGYTFTDYYINWVKQKPEQGLEWIGWTFPGSINTKYNEKFKGKATLTVDTSSSTAYMQLNSLTSEDTAVYFCARSPYEFDYWGQGTTLTVSS >PDL01043_3G11LH-SEQ ID NO: 263 DIQMTQTTSSLSASLGDRVTISCSASQGIRNYLNWYQQKPDGTVKLLIYYTSTLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPWTFGGGTRLEIKGGGGSGGGGSGGGGSG GGGSEVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGKKLEYMGYISYTGSTYYNPSLKSRISIARDTSKNQYFLQLNSVTTEDTATYYCTRGENWDGGYFDYWGQGTTLTVSS >PDL01045_6F6LH-SEQ ID NO: 264 DIVMTQSHKFMSTSVGDRVTITCKASQDVGTAVAWYQKKPGQSPKLLISWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGGGTNLEIKGGGGSGGGGSGGGGSG GGGSQGQMQQSGAELVKPGASVKLSCKTSGFTFSSSYITWLKQKPGQSLEWIAWIYVGTGGISYNQKFTGKAQLTVDTSSSTAYMQFSSLTTEDSAIYYCARHGRYHWYFDVWGAGTTVTVSS >PDL01047_7A3-2LH-SEQ ID NO: 265 DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPFTFGAGTKLEIKGGGGSGGGGSGGGGGSGG GGSEVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNRLEYMGFRSYSGTTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCARFSDFGSSENTMDYWGQGTSVTVSS >PDL01048_7H11LH-SEQ ID NO: 266 DIVLTQSPASLAVSLGQRATISCRASKSVSKSGYSYLHWYQQKPGQTPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSWELPYTFGGGTKLEIKGGGGSGGGGSGGGG SGGGGSEVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGKKLEYMGYISYTGSTYYNPSLKSRISIARDTSKNQYFLQLNSVTTEDTATYYCTRGENWDGGYFDYWGQGTTLTVSS >PDL01049_10B2LH-SEQ ID NO: 267 DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLQSGVPSRFSGSGSGTDYSLTITTLEQEDFATYFCQQGNMFPYTFGGGTKLEIKGGGGSGGGGSGGGS GGGGSKVQLQQSGAEVVKPGASVKLSCKASGYNLTEYIIHWVKQRSGQGLEWIGWFYPGSGSIKYNEKFKDKATLTADKSSTTVYMDLSRLTSEDSAVYFCARHGLYYGFPYWGQGTSVTVSS >PDL01050_Light_Chain_5F11_Chimeric_mAb-SEQ ID NO: 268 DIQMTQSPSSLSASLGGKVTITCKASQDINKYIAWYQHKPGKGPRLLIHYTSILQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLRTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >PDL01050_heavy chain_5F11 chimeric_mAb-SEQ ID NO: 269 EVKFEESGGGLVQPGGSMKLSCFASGFTFSYYWMNWVRQSPEKGLEWIAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLEMNNLRAEDTGIYHCTRPPIYYGNYEAYWG QGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >PDL01051_Light Chain_7A3-1 Chimeric_mAb-SEQ ID NO: 270 DIVMTQSHKFMSTSVGDRVTITCKASQDVGTAVAWYQKKPGQSPKLLISWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGGGTNLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >PDL01051_Heavy Chain_7A3-1 Chimeric mAb-SEQ ID NO: 271 EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNRLEYMGFRSYSGTTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCARFSDFGSSENTMDYWG QGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >PC401025-SEQ ID NO: 272 DIQMTQSPSSLSASVGDRVTINCQASQSIDSNLAWFQQKPGQPPKLLIYRASNLASGVPDRFSGSGSGTDFTLTISSLEAEDVATYYCLGGVGAVSYRTSFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCTASGSDINDYPITWVRQAPGQGLEWIGFINSGG STWYASWVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARGYSTYYRDFNIWGQGTLVTVSSSEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSDIQMTQSPSSVSASVGDRVTITCRASQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASSLQSGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWIEWIRQAPGQGLEWMGSILPGSGSTNYAQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCARGDAGSYVRWYFDHWGQGTLVTVSS >PC401027-SEQ ID NO: 273 DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGGGGSGGGGSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNT PFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSSEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSDIQMTQSPSSVSASVGDRVTITCRASQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASSLQSGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWIEWIRQAPGQGLEWMGSILPGSGSTNYAQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCARGDAGSYVRWYFDHWGQGTLVTVSS >PC401035-SEQ ID NO: 274 DIQMTQSPSSLSASVGDRVTINCQASQSIDSNLAWFQQKPGQPPKLLIYRASNLASGVPDRFSGSGSGTDFTLTISSLEAEDVATYYCLGGVGAVSYRTSFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCTASGSDINDYPITWVRQAPGQGLEWIGFINSGG STWYASWVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARGYSTYYRDFNIWGQGTLVTVSSSEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSDIQMTQSPSSVSASVGDRVTITCRASQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASSLQSGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWIEWIRQAPGQGLEWMGSILPGSGSTNYAQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >PC401037-SEQ ID NO: 275 DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGGGGSGGGGSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNT PFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSSEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSDIQMTQSPSSVSASVGDRVTITCRASQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASSLQSGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWIEWIRQAPGQGLEWMGSILPGSGSTNYAQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >PC401015-SEQ ID NO: 490 DIQMTQSPSSLSASVGDRVTITCRASQDINKFLSWYQQKPGKAPKLLIHYTSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDNLYTFGQGTKLEIKGGGGGSGGGGSGGG GSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVSFITSLAYIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARNYGSSPYYFDYWGQ GTLVTVSSEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >CD4001272-SEQ ID NO: 491 EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSDIQMTQSPS SVSASVGDRVTITCRASQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGGGSGGGG SQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWIEWIRQAPGQGLEWMGSILPGSGSTNYAQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001242-SEQ ID NO: 492 EPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSPSDIQMTQSPS SVSASVGDRVTITCRASQSLLFSVNQKNYLAWYQQKPGKAPKLLIYWASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYSYPWTFGQGTKVEIKGGGGSGGGGSGGGGGSGGGG SQVQLVQSGAEVKKPGSSVKVSCKASGYTFSTSWIEWIRQAPGQGLEWMGSILPGSGSTNYAQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCARGDDGSYVRWYFDHWGQGTLVTVSS >CD4001085_light chain—SEQ ID NO: 493 DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >CD4001085_heavy chain—SEQ ID NO: 494 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYF DYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEK TISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0091] Provided herein is a CD40-binding polypeptide that specifically binds to human CD40, comprising, in amino-terminal to carboxyl-terminal or carboxyl-terminal to amino-terminal order: (a) a first binding domain, (b) a hinge region, (c) an immunoglobulin constant region, and (d) a second binding domain, wherein the first binding domain is a CD40-binding domain and the second binding domain binds to a tumor-associated antigen, or the first binding domain binds to a tumor-associated antigen and the second binding domain is a CD40-binding domain. The first binding domain, the second binding domain, or both binding domains can be scFvs. In some embodiments, the tumor-associated antigen is PD-L1, ROR1, or EGFR.

[0092] In some embodiments, the CD40 binding domain comprises (i) an immunoglobulin heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3, and (ii) an immunoglobulin light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3. Exemplary combinations of regions of CD40 binding domain sequences are shown in Table 3.

[0093] In some embodiments, (a) HCDR1 comprises SEQ ID NO: 10, HCDR2 comprises SEQ ID NO: 11, HCDR3 comprises SEQ ID NO: 12, LCDR1 comprises SEQ ID NO: 14, LCDR2 comprises SEQ ID NO: 15, and LCDR3 comprises SEQ ID NO: 16; (b) HCDR1 comprises SEQ ID NO: 10, HCDR2 comprises SEQ ID NO: 23, HCDR3 comprises SEQ ID NO: 12, LCDR1 comprises SEQ ID NO: 14, LCDR2 comprises SEQ ID NO: 15, and LCDR3 comprises SEQ ID NO: 16; or (c) HCDR1 comprises SEQ ID NO: 10. and HCDR2 comprises SEQ ID NO: 11, HCDR3 comprises SEQ ID NO: 31, LCDR1 comprises SEQ ID NO: 14, LCDR2 comprises SEQ ID NO: 15, and LCDR3 comprises SEQ ID NO: 16; (d) HCDR1 comprises SEQ ID NO: 10, HCDR2 comprises SEQ ID NO: 23, and HCDR3 comprises SEQ ID NO: 31, LCDR1 comprises SEQ ID NO: 14, LCDR2 comprises SEQ ID NO: 15, and LCDR3 comprises SEQ ID NO: 16; or (e) HCDR1 comprises SEQ ID NO: 10, HCDR2 comprises SEQ ID NO: 11, and HCDR3 comprises SEQ ID NO: 12, wherein LCDR1 comprises SEQ ID NO:37, LCDR2 comprises SEQ ID NO:15, and LCDR3 comprises SEQ ID NO:16; (f) HCDR1 comprises SEQ ID NO:10, HCDR2 comprises SEQ ID NO:11, and HCDR3 comprises SEQ ID NO:12, wherein LCDR1 comprises SEQ ID NO:41, LCDR2 comprises SEQ ID NO:15, and LCDR3 comprises SEQ ID NO:16; (g) HCDR1 comprises SEQ ID NO:10, HCDR2 comprises SEQ ID NO:45, and HCDR3 comprises SEQ ID NO:12, wherein LCDR1 comprises SEQ ID NO:14, and LCDR2 comprises (h) HCDR1 comprises SEQ ID NO: 10, HCDR2 comprises SEQ ID NO: 49, HCDR3 comprises SEQ ID NO: 12, LCDR1 comprises SEQ ID NO: 14, LCDR2 comprises SEQ ID NO: 15, and LCDR3 comprises SEQ ID NO: 16, or (i) HCDR1 comprises SEQ ID NO: 10, HCDR2 comprises SEQ ID NO: 23, HCDR3 comprises SEQ ID NO: 31, LCDR1 comprises SEQ ID NO: 37, LCDR2 comprises SEQ ID NO: 15, and LCDR3 comprises SEQ ID NO: 16.

[0094] In some embodiments, (a) the VH comprises SEQ ID NO: 13 and the VL comprises SEQ ID NO: 17; (b) the VH comprises SEQ ID NO: 24 and the VL comprises SEQ ID NO: 17; (c) the VH comprises SEQ ID NO: 27 and the VL comprises SEQ ID NO: 28; (d) the VH comprises SEQ ID NO: 32 and the VL comprises SEQ ID NO: 17; (e) the VH comprises SEQ ID NO: 34 and the VL comprises SEQ ID NO: 17; or (f) the VH comprises SEQ ID NO: 1 3 and the VL comprises SEQ ID NO: 38; (g) the VH comprises SEQ ID NO: 13 and the VL comprises SEQ ID NO: 42; (h) the VH comprises SEQ ID NO: 46 and the VL comprises SEQ ID NO: 17; (i) the VH comprises SEQ ID NO: 50 and the VL comprises SEQ ID NO: 17; (j) the VH comprises SEQ ID NO: 484 and the VL comprises SEQ ID NO: 485; or (k) the VH comprises SEQ ID NO: 484 and the VL comprises SEQ ID NO: 28.

[0095] In some embodiments, the CD40 binding domain comprises SEQ ID NO: 18, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 486, or SEQ ID NO: 488. In some embodiments, the CD40 binding domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NO: 18, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 486, and SEQ ID NO: 488.

[0096] In some embodiments, the second binding domain comprises (i) an immunoglobulin heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3, and (ii) an immunoglobulin light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3. Table 3. SEQ ID NOs of CD40 binding domain sequence regions [Table 2]

[0097] Provided herein are binding polypeptides that specifically bind to human PD-L1 and human CD40. Provided herein are binding polypeptides that specifically bind to human PD-L1 and human CD40, comprising, in order from amino terminus to carboxyl terminus, or from carboxyl terminus to amino terminus, (a) a first binding domain, (b) a hinge region, (c) an immunoglobulin constant region, and (d) a second binding domain, wherein the first binding domain is a PD-L1-binding domain and the second binding domain is a CD40-binding domain, or the first binding domain is a CD40-binding domain and the second binding domain is a PD-L1-binding domain.

[0098] Provided herein is a binding polypeptide that specifically binds to human PD-L1 and human CD40, the binding polypeptide comprising, in order from amino terminus to carboxyl terminus: (a) a first binding domain, (b) a hinge region, (c) an immunoglobulin constant region, and (d) a second binding domain, wherein the first binding domain is a PD-L1-binding domain and the second binding domain is a CD40-binding domain.

[0099] The CD40 binding domain, the PD-L1 binding domain, or both binding domains can be scFvs.

[0100] Provided herein are binding polypeptides that specifically bind to human PD-L1 and human CD40, the binding polypeptides comprising, in order from amino terminus to carboxyl terminus, (i) a first binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) an Fc binding domain, and (v) a second binding domain, wherein the first binding domain is a PD-L1 binding domain and the second binding domain is a CD40 binding domain. In some embodiments, the Fc binding domain linker is GGGGSPS (SEQ ID NO: 21).

[0101] Provided herein is a binding polypeptide that specifically binds to human PD-L1 and human CD40, the binding polypeptide comprising, in order from amino terminus to carboxyl terminus, (i) a PD-L1-binding domain, (ii) an Fc region, and (iii) a CD40-binding domain. In some embodiments, the Fc region comprises or consists of SEQ ID NO:22.

[0102] Provided herein is a binding polypeptide, wherein the PD-L1-binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; and the CD40-binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 11, and an HCDR3 comprising SEQ ID NO: 12, and a VL comprising an LCDR1 comprising SEQ ID NO: 14, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO: 16.

[0103] Provided herein is a binding polypeptide, wherein the PD-L1-binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; and the CD40-binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 23, and an HCDR3 comprising SEQ ID NO: 12, and a VL comprising an LCDR1 comprising SEQ ID NO: 14, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO: 16.

[0104] Provided herein is a binding polypeptide, wherein the PD-L1-binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; and the CD40-binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 11, and an HCDR3 comprising SEQ ID NO: 31, and a VL comprising an LCDR1 comprising SEQ ID NO: 14, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO: 16.

[0105] Provided herein is a binding polypeptide, wherein the PD-L1-binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 7, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; and the CD40-binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 23, and an HCDR3 comprising SEQ ID NO: 31, and a VL comprising an LCDR1 comprising SEQ ID NO: 14, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO: 16.

[0106] Provided herein is a binding polypeptide, wherein the PD-L1-binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; and the CD40-binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 11, and an HCDR3 comprising SEQ ID NO: 12, and a VL comprising an LCDR1 comprising SEQ ID NO: 37, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO: 16.

[0107] Provided herein is a binding polypeptide, wherein the PD-L1-binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; and the CD40-binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 11, and an HCDR3 comprising SEQ ID NO: 12, and a VL comprising an LCDR1 comprising SEQ ID NO: 41, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO: 16.

[0108] Provided herein is a binding polypeptide, wherein the PD-L1-binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; and the CD40-binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 45, and an HCDR3 comprising SEQ ID NO: 12, and a VL comprising an LCDR1 comprising SEQ ID NO: 14, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO: 16.

[0109] Provided herein is a binding polypeptide, wherein the PD-L1-binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; and the CD40-binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 49, and an HCDR3 comprising SEQ ID NO: 12, and a VL comprising an LCDR1 comprising SEQ ID NO: 14, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO: 16.

[0110] Provided herein is a binding polypeptide, wherein the PD-L1-binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; and the CD40-binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 23, and an HCDR3 comprising SEQ ID NO: 31, and a VL comprising an LCDR1 comprising SEQ ID NO: 37, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO: 16.

[0111] Provided herein is a binding polypeptide, wherein (a) the PD-L1-binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40-binding domain comprises a VH comprising SEQ ID NO: 13 and a VL comprising SEQ ID NO: 17; (b) the PD-L1-binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40-binding domain comprises a VH comprising SEQ ID NO: 24 and a VL comprising SEQ ID NO: 17; or (c) the PD-L1-binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40-binding domain comprises a VH comprising SEQ ID NO: 24 and a VL comprising SEQ ID NO: 17. (d) the PD-L1 binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40 binding domain comprises a VH comprising SEQ ID NO: 32 and a VL comprising SEQ ID NO: 17; (e) the PD-L1 binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40 binding domain comprises a VH comprising SEQ ID NO: 34 and a VL comprising SEQ ID NO: 17; or (f) the PD-L1 binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8. (g) the PD-L1 binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40 binding domain comprises a VH comprising SEQ ID NO: 13 and a VL comprising SEQ ID NO: 38; (g) the PD-L1 binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40 binding domain comprises a VH comprising SEQ ID NO: 13 and a VL comprising SEQ ID NO: 42; (h) the PD-L1 binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40 binding domain comprises a VH comprising SEQ ID NO: 46 and a VL comprising SEQ ID NO: 17; or (i) the PD-L1 binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: (j) the PD-L1 binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40 binding domain comprises a VH comprising SEQ ID NO: 50 and a VL comprising SEQ ID NO: 17; or (k) the PD-L1 binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40 binding domain comprises a VH comprising SEQ ID NO: 484 and a VL comprising SEQ ID NO: 28.

[0112] Provided herein is a binding polypeptide, wherein (a) the PD-L1-binding domain comprises SEQ ID NO: 9 and the CD40-binding domain comprises SEQ ID NO: 18; (b) the PD-L1-binding domain comprises SEQ ID NO: 9 and the CD40-binding domain comprises SEQ ID NO: 25; (c) the PD-L1-binding domain comprises SEQ ID NO: 9 and the CD40-binding domain comprises SEQ ID NO: 29; (d) the PD-L1-binding domain comprises SEQ ID NO: 9 and the CD40-binding domain comprises SEQ ID NO: 33; (e) the PD-L1-binding domain comprises SEQ ID NO: 9 and the CD40-binding domain comprises SEQ ID NO: 35; and (f) the PD-L1-binding domain comprises SEQ ID NO: 9. (g) the PD-L1 binding domain comprises SEQ ID NO: 9 and the CD40 binding domain comprises SEQ ID NO: 43; (h) the PD-L1 binding domain comprises SEQ ID NO: 9 and the CD40 binding domain comprises SEQ ID NO: 47; (i) the PD-L1 binding domain comprises SEQ ID NO: 9 and the CD40 binding domain comprises SEQ ID NO: 51; (j) the PD-L1 binding domain comprises SEQ ID NO: 9 and the CD40 binding domain comprises SEQ ID NO: 486; and (k) the PD-L1 binding domain comprises SEQ ID NO: 9 and the CD40 binding domain comprises SEQ ID NO: 488.

[0113] Provided herein are binding polypeptides comprising SEQ ID NO: 19, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 280, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 487, or SEQ ID NO: 489. Provided herein are binding polypeptides comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NO: 19, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 280, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 487, and SEQ ID NO: 489.

[0114] Provided herein are binding polypeptides comprising the amino acid sequence of the molecule PC401003, and its variants PC401119, PC401120, PC401122, PC401124, PC401128, PC401129, PC401132, PC401133, PC401144, or PC401145.

[0115] Compared to PC401003, PC401119 contains an A to N mutation in anti-CD40 HCDR2, PC401120 contains a Q to C mutation in anti-CD40 LFW4 and a G to C mutation in anti-CD40 HFW2, PC401122 contains a V to N mutation in anti-CD40 HCDR3, PC401124 contains an A to N mutation in anti-CD40 HCDR2 and a V to N mutation in anti-CD40 HCDR3, PC401128 contains an L to T mutation in anti-CD40 LCDR1, PC401129 contains an L to N mutation in anti-CD40 LCDR1, PC401132 contains an L to N mutation in anti-CD40 HCDR2, and PC401133 contains an L to N mutation in anti-CD40 PC401144 contains an L to Q mutation in HCDR2, an A to N mutation in anti-CD40 HCDR2, a V to N mutation in anti-CD40 HCDR3, and an L to T mutation in anti-CD40 LCDR1, and PC401145 contains an A to N mutation in anti-CD40 HCDR2 and a V to N mutation in anti-CD40 HCDR3. These amino acid substitution mutations are shown in Table 1.

[0116] Provided herein are binding polypeptides comprising a CD40 binding domain, wherein the CD40 binding domain is a variant of the PC401003 CD40 binding domain that includes two, three, four, five, six, or seven of the amino acid substitutions found in PC401119, PC401120, PC401122, PC401124, PC401128, PC401129, PC401132, PC401133, PC401144, or PC401145.

[0117] The binding domains described herein may comprise or consist of a single chain variable fragment (scFv). In some embodiments, the scFv comprises a V H -V L Direction or V L -V H In some embodiments, the VH may be carboxy-terminal to the VL. In some embodiments, the VL may be carboxy-terminal to the VH.

[0118] In some embodiments, the scFv is H Area and V L A linker (i.e., an scFv linker) may be included between the regions. In some embodiments, the linker may comprise GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 20).

[0119] The binding polypeptides provided herein can comprise an immunoglobulin Fc (Fc) region. The Fc region described herein can comprise an IgG CH2 and CH3 domain, e.g., the CH2 and CH3 domains of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD. In certain embodiments, the Fc region does not comprise a CH1 domain. In certain embodiments, the sequences comprising the Fc region are human or derived from human sequences. In some embodiments, the Fc region comprises a human IgG1 CH2 domain comprising an amino acid substitution at one or more of the following residues according to the EU numbering system: E233, L234, L235, G236, G237, E318, K320, and K322. In some embodiments, the amino acid substitution at residue E233 is E233P. In some embodiments, the amino acid substitution at residue L234 is selected from the group consisting of L234A and L234V. In some embodiments, the amino acid substitution at residue L235 is L235A. In some embodiments, the amino acid substitution at residue G237 is G237A. In some embodiments, the amino acid substitution at E318 is E318A. In some embodiments, the amino acid substitution at K320 is K320A. In some embodiments, the amino acid substitution at K322 is K322A. In some embodiments, one or more of E233, L234, L235, G236, G237, E318, K320, and K322 are deleted. In certain embodiments, residue G236 is deleted.

[0120] In some embodiments, the Fc domain is derived from IgG1. In some embodiments, the Fc domain derived from IgG1 comprises two or more mutations that prevent the polypeptide from depleting PD-L1 and / or CD40-expressing cells when administered to a patient. In some embodiments, the two or more mutations in the IgG1 Fc domain prevent or substantially reduce signaling via Fc-mediated cross-linking.

[0121] In some embodiments, the Fc comprises SEQ ID NO:22.

[0122] In some embodiments, the binding polypeptide comprises a hinge that is an immunoglobulin hinge. The hinge region described herein can be derived from IgG. In some embodiments, the hinge region has one or more mutated cysteine ​​residues.

[0123] In some embodiments, the binding polypeptides provided herein are single-chain polypeptides.

[0124] In some embodiments, the binding polypeptide is a dimer, e.g., a homodimer or a heterodimer. In some embodiments, the dimeric polypeptide comprises at least one binding polypeptide of the present disclosure. In some embodiments, the dimeric polypeptide comprises two binding polypeptides of the present disclosure. In some embodiments, the binding polypeptides of the present disclosure can be dimerized into identical binding polypeptides. In some embodiments, the bispecific single-chain binding polypeptide exists predominantly in a homodimeric form. In some embodiments, the binding polypeptides of the present disclosure can be dimerized into a non-identical second polypeptide.

[0125] Polynucleotide and protein expression methods The present disclosure also includes nucleic acids (e.g., DNA or RNA) encoding one or more polypeptide chains of a polypeptide of the present disclosure (e.g., a bispecific PD-L1-binding polypeptide, a bispecific CD40-binding polypeptide, or a bispecific PD-L1 x CD40-binding polypeptide) or a polypeptide described herein. Nucleic acids of the present disclosure also include complementary nucleic acids. In some cases, the sequences are perfectly complementary (no mismatches) when aligned. In other cases, there may be up to about 20% mismatches in the sequences. In some embodiments of the present disclosure, nucleic acids are provided that encode both the first and second polypeptide chains of a bispecific protein of the present disclosure. The nucleic acid sequences provided herein may be utilized to optimize expression in a particular host using codon optimization, degenerate sequences, silent mutations, and other DNA techniques, and the present disclosure encompasses such sequence modifications.

[0126] The present disclosure relates to isolated nucleic acid molecules encoding the polypeptides of the disclosure (e.g., PD-L1-binding domains, CD40-binding domains, and bispecific PD-L1xCD40-binding polypeptides). Provided herein are nucleic acid molecules comprising a nucleotide sequence comprising SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:259, or SEQ ID NO:260.

[0127] Polynucleotide molecules containing a desired polynucleotide sequence are propagated by placing the molecule in a vector. Viral and non-viral vectors are used. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentivirus, retrovirus, adenovirus, or adeno-associated virus (AAV). In some embodiments, the vectors of the present disclosure are plasmids. The choice of plasmid depends on the type of cell desired to be propagated and the purpose of propagation. Certain vectors are useful for amplifying and producing large amounts of the desired DNA sequence. Other vectors are suitable for expression in cells in culture. Still other vectors are suitable for transfer and expression in whole animal or human cells. Selection of an appropriate vector is well within the skill of one in the art. Many such vectors are commercially available. A partial or full-length polynucleotide is typically inserted into the vector by binding of DNA ligase to a restriction enzyme site cleaved within the vector. Alternatively, the desired nucleotide sequence can be inserted by homologous recombination in vivo. Typically, this is accomplished by flanking the desired nucleotide sequence with regions of homology to the vector, which are added, for example, by ligation of oligonucleotides or by polymerase chain reaction using primers that contain both the regions of homology and a portion of the desired nucleotide sequence.

[0128] For expression, an expression cassette or expression system may be used. To express a nucleic acid encoding a polypeptide disclosed herein, a nucleic acid molecule encoding the polypeptide, operably linked to regulatory sequences controlling transcriptional expression in an expression vector, is introduced into a host cell. Transcriptional regulatory sequences may include a promoter and / or enhancer, and the promoter may be constitutive or inducible. In addition to transcriptional regulatory sequences, the expression vector may also include translational regulatory sequences and a marker gene suitable for selection of cells harboring the expression vector. Gene products encoded by the polynucleotides of the present disclosure may be expressed in any convenient expression system, including, for example, bacterial, yeast, insect, amphibian, and mammalian systems. Within the expression vector, the polypeptide-encoding polynucleotide is optionally linked to regulatory sequences to obtain the desired expression characteristics. These may include promoters, enhancers, terminators, operators, repressors, and inducers. Promoters can be regulated (e.g., promoters from the steroid-inducible pIND vector (Invitrogen)) or constitutive (e.g., promoters from CMV, SV40, elongation factors, or LTR sequences). These are ligated to the desired nucleotide sequence using the techniques described above for ligation into vectors. Any technique known in the art can be used. Thus, an expression vector generally provides transcription and translation initiation regions, which can be inducible or constitutive, a coding region operably linked under the transcriptional control of the transcription initiation region, and has transcription and translation termination regions.

[0129] The vectors described herein can be contained within a host cell, such as a CHO cell or an HEK cell. In some embodiments, the host cell is stably transfected with the vector, e.g., a stably transfected CHO or HEK cell.

[0130] Expression cassettes ("expression units") can be introduced into a variety of vectors, such as plasmids, BACs, YACs, bacteriophages such as lambda, P1, and M13, plant or animal viral vectors (e.g., retroviral-based vectors, adenoviral vectors), and the vectors are typically characterized by their ability to provide for selection of cells containing the expression vector. Vectors can provide for extrachromosomal maintenance, particularly as plasmids or viruses, or for integration into host chromosomes. If extrachromosomal maintenance is desired, an origin sequence for replication of the plasmid is provided, which can be low or high copy number. A wide variety of markers are available for selection, including markers that protect against toxins, more specifically antibiotics. The particular marker selected will depend on the nature of the host; in some cases, complementation can be used in auxotrophic hosts. Any convenient method can be used to introduce the DNA construct, including, for example, conjugation, bacterial transformation, calcium-precipitated DNA, electroporation, fusion, transfection, infection with viral vectors, biolistics, and the like.

[0131] Thus, proteins for use within the present disclosure can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are cell types that can be transformed or transfected with exogenous DNA and grown in culture, including bacteria, fungal cells, and cultured higher eukaryotic cells (including cultured cells of multicellular organisms), particularly cultured mammalian cells. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into various host cells are disclosed by Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001), and Ausubel et al., Short Protocols in Molecular Biology (4th ed., John Wiley & Sons, 1999).

[0132] For example, in the case of recombinant expression of a homodimeric binding protein comprising two identical binding polypeptides as described herein, an expression vector generally comprises a nucleic acid segment encoding the binding polypeptide operably linked to a promoter. In the recombinant expression of a heterodimeric binding protein comprising different first and second polypeptide chains, the first and second polypeptide chains may be co-expressed from separate vectors in a host cell to express the entire heterodimeric protein. Alternatively, in the recombinant expression of a heterodimeric binding protein, the first and second polypeptide chains are co-expressed from separate expression units in the same vector in a host cell to express the entire heterodimeric protein. The expression vector(s) are transferred into host cells by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the encoded polypeptide(s) to produce the corresponding binding protein (e.g., a PD-L1xCD40 polypeptide).

[0133] A secretory signal sequence (also known as a leader sequence) is provided in the expression vector to direct the recombinant protein into the secretory pathway of the host cell. The secretory signal sequence can be that native to the recombinant protein, or it can be derived from another secreted protein, or it can be synthesized de novo. The secretory signal sequence is operably linked to the DNA sequence encoding the polypeptide; i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences can be positioned elsewhere in the DNA sequence of interest (see, e.g., U.S. Pat. Nos. 5,037,743 and 5,143,830).

[0134] Cultured mammalian cells are suitable hosts for production of the recombinant polypeptides and proteins of the present disclosure (e.g., PD-L1×CD40 polypeptides) for use within the present disclosure. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran-mediated transfection (Ausubel et al., supra), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993). The production of recombinant polypeptides in cultured mammalian cells is disclosed in, for example, US Pat. Nos. 4,713,339, 4,784,950, 4,579,821, and 4,656,134. Examples of suitable mammalian host cells include African green monkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1; ATCC CCL61; CHO DG44; CHO DXB11 (Hyclone, Logan, UT); see also, e.g., Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986), rat pituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548), SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650), and mouse embryonic cells (NIH-3T3; ATCC CRL 1658).Additional suitable cell lines are known in the art and are available from public depositories such as the American Type Culture Collection, Manassas, Virginia. Strong transcription promoters, such as promoters from SV-40 or cytomegalovirus, can be used. See, e.g., U.S. Patent No. 4,956,288. Other suitable promoters include those derived from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

[0135] Drug selection is commonly used to select cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants." Cells that can be cultured in the presence of a selective drug and pass on the gene of interest to their progeny are called "stable transfectants." Exemplary selectable markers include genes encoding resistance to the antibiotic neomycin (allowing selection in the presence of neomycin-type drugs such as G-418), the gpt gene for xanthine-guanine phosphoribosyltransferase (allowing host cells to grow in the presence of mycophenolic acid / xanthine), and markers that confer resistance to zeocin, bleomycin, blastocidin, and hygromycin (see, e.g., Gatignol et al., Mol. Gen. Genet. 207:342, 1987; Drocourt et al., Nucl. Acids Res. 18:4009, 1990). Selection systems can also be used to increase the expression level of the gene of interest, a process known as "amplification." Amplification is achieved by culturing transfectants in the presence of low levels of a selection agent, then increasing the amount of selection agent to select for cells that produce high levels of the transgene product. An exemplary amplifiable selection marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (e.g., hygromycin resistance, multidrug resistance, puromycin acetyltransferase) can also be used.

[0136] Other higher eukaryotic cells, including insect cells, plant cells, and avian cells, can also be used as hosts. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells is reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed in U.S. Patent No. 5,162,222 and PCT Publication No. WO 94 / 06463.

[0137] Insect cells can be infected with recombinant baculoviruses, typically derived from Autographa californica nuclear polyhedrosis virus (AcNPV). See King and Possee, The Baculovirus Expression System: A Laboratory Guide (Chapman & Hall, London); O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual (Oxford University Press, New York 1994); and Baculovirus Expression Protocols. Methods in Molecular Biology (Richardson ed., Humana Press, Totowa, NJ, 1995). Recombinant baculoviruses can also be produced through the use of the transposon-based system described by Luckow et al. (J. Virol. 67:4566-4579, 1993). This system, which utilizes transfer vectors, is commercially available in kit form (BAC-TO-BAC kit; Life Technologies, Gaithersburg, MD). Transfer vectors (e.g., PFASTBAC1; Life Technologies) contain a Tn7 transposon for moving DNA encoding a protein of interest into the baculovirus genome, which is maintained in E. coli as a large plasmid called a "bacmid." See Hill-Perkins and Possee, J. Gen. Virol. 71:971-976, 1990; Bonning et al., J. Gen. Virol. 75:1551-1556, 1994; and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543-1549, 1995. Additionally, transfer vectors can contain in-frame fusions with DNA encoding the polypeptide extensions or affinity tags disclosed above.Using techniques known in the art, a transfer vector containing a DNA sequence encoding the protein is transformed into E. coli host cells, and the cells are screened for bacmids containing an interrupted lacZ gene, indicating a recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus genome is isolated using common techniques and used to transfect Spodoptera frugiperda cells, such as Sf9 cells. Recombinant viruses expressing the protein of interest are then produced. Recombinant virus stocks are generated using methods commonly used in the art.

[0138] For protein production, recombinant viruses are used to infect host cells, typically cell lines derived from the fall armyworm, Spodoptera frugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., HIGH FIVE™ cells; Invitrogen, Carlsbad, CA). See generally Glick and Pasternak, Molecular Biotechnology, Principles & Applications of Recombinant DNA (ASM Press, Washington, DC, 1994). See also U.S. Pat. No. 5,300,435. Serum-free medium is used to grow and maintain the cells. Suitable medium compositions are known in the art and available from commercial suppliers. Cells are grown at approximately 2-5 x 10 5 Cell seeding density ranges from 1 to 2 x 10 6 Cells are grown to density, at which point recombinant virus stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically closer to 3. Procedures for use are generally described in available laboratory manuals (see, e.g., King and Possee, supra; O'Reilly et al., supra; Richardson, supra).

[0139] Fungal cells, including yeast cells, may also be used within the present disclosure to produce polypeptides of the present disclosure (e.g., PD-L1×CD40 polypeptides). Yeast species in this regard include, for example, Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed, for example, in U.S. Patent Nos. 4,599,311, 4,931,373, 4,870,008, 5,037,743, and 4,845,075. Transformed cells are selected by a phenotype determined by a selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). An exemplary vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformed cells to be selected by growth on glucose-containing medium. Suitable promoters and terminators for use in yeast include those derived from glycolytic enzyme genes (see, e.g., U.S. Pat. Nos. 4,599,311, 4,615,974, and 4,977,092) and alcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446, 5,063,154, 5,139,936, and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii, and Candida maltosa, are known in the art.See, e.g., Gleeson et al., J. Gen. Microbiol. 132:3459-3465, 1986; U.S. Pat. No. 4,882,279; and Raymond et al., Yeast 14:11-23, 1998. Aspergillus cells can be utilized according to the method of McKnight et al., U.S. Pat. No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Pat. No. 4,486,533. Production of recombinant proteins in Pichia methanolica is disclosed in U.S. Pat. Nos. 5,716,808, 5,736,383, 5,854,039, and 5,888,768.

[0140] Prokaryotic host cells, including strains of the bacteria Escherichia coli, Bacillus, and other genera, are also useful host cells within the present disclosure for producing multispecific binding proteins, including, for example, PD-L1-binding polypeptides, CD40-binding polypeptides, and / or PDL1×CD40-binding polypeptides. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook and Russell, supra). When recombinant proteins are expressed in bacteria such as E. coli, the protein can typically be retained in the cytoplasm as insoluble granules or can be targeted to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured, for example, using guanidine isothiocyanate or urea. The denatured protein can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against buffered saline. Alternatively, proteins can be recovered from the cytoplasm in a soluble form and isolated without the use of denaturants. Proteins are recovered from cells, for example, as an aqueous extract in phosphate-buffered saline. To capture the protein of interest, the extract is applied directly to a chromatography medium, such as an immobilized antibody or heparin-Sepharose column. Secreted proteins can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (e.g., by sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby eliminating the need for denaturation and refolding. Antibodies, including single-chain antibodies, can be produced in bacterial host cells according to known methods. See, e.g., Bird et al., Science 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; and Pantoliano et al., Biochem. 30:10117-10125, 1991.

[0141] Host cells transformed or transfected to produce the polypeptides and proteins of the present disclosure (e.g., PD-L1-binding polypeptides, CD40-binding polypeptides, or PD-L1×CD40 polypeptides) are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the selected host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins, and minerals. The medium may also include components such as growth factors or serum, as needed. The growth medium generally selects for cells containing the exogenously added DNA, for example, by drug selection or by deficiency of essential nutrients complemented by a selectable marker carried on the expression vector or co-transfected into the host cells.

[0142] Proteins and polypeptides (e.g., PD-L1×CD40 polypeptides) of the disclosure may be purified by conventional protein purification methods, typically by a combination of chromatographic techniques. See generally, Affinity Chromatography: Principles & Methods (Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988); Scopes, Protein Purification: Principles and Practice (Springer-Verlag, New York 1994). Proteins containing immunoglobulin Fc regions may be purified by affinity chromatography on immobilized Protein A or Protein G. Additional purification steps, such as gel filtration, may be used to obtain the desired level of purity, or to perform desalting, buffer exchange, etc.

[0143] Compositions and Methods of Use For administration to a subject, proteins of the present disclosure (e.g., bispecific PD-L1-binding polypeptides, bispecific CD40-binding polypeptides, or bispecific PD-L1×CD40-binding polypeptides, or dimers thereof) may be formulated as pharmaceutical compositions. Pharmaceutical compositions may comprise (i) binding polypeptides of the present disclosure and / or dimers thereof, and (ii) a pharmaceutically acceptable carrier, diluent, or excipient. Pharmaceutical compositions comprising binding polypeptides of the present disclosure may be formulated according to known methods for preparing pharmaceutically useful compositions, in which a therapeutic molecule is combined in a mixture with a pharmaceutically acceptable carrier, diluent, or excipient. A carrier is said to be a "pharmaceutically acceptable carrier" if its administration can be tolerated by a recipient patient. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers, diluents, or excipients will be known to those of skill in the art. (See, e.g., Gennaro (ed.), Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995).) The formulation may further include one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss, such as on the vial surface. In certain embodiments, the pharmaceutical composition comprises a bispecific PD-L1×CD40 binding polypeptide that is a homodimer or a heterodimer. A "homodimer" may be a dimer formed from two identical polypeptides.

[0144] Thus, in some embodiments, a pharmaceutical composition may comprise a PD-L1 binding polypeptide, a bispecific CD40 binding polypeptide, or a bispecific PD-L1xCD40 binding polypeptide of the disclosure dimerized to the same binding polypeptide, and a pharmaceutically acceptable buffer or excipient.

[0145] Pharmaceutical compositions comprising a bispecific PD-L1-binding polypeptide, a bispecific CD40-binding polypeptide, or a bispecific PD-L1×CD40-binding polypeptide of the present disclosure may be formulated into a dosage form selected from the group consisting of: oral unit dosage form, intravenous unit dosage form, intranasal unit dosage form, suppository unit dosage form, intradermal unit dosage form, intramuscular unit dosage form, intraperitoneal unit dosage form, subcutaneous unit dosage form, epidural unit dosage form, sublingual unit dosage form, and intracerebral unit dosage form. Oral unit dosage forms may be selected from the group consisting of tablets, pills, pellets, capsules, powders, lozenges, granules, solutions, suspensions, emulsions, syrups, elixirs, sustained release formulations, aerosols, and sprays.

[0146] Pharmaceutical compositions comprising a polypeptide or protein described herein (e.g., a bispecific PD-L1-binding polypeptide, a bispecific CD40-binding polypeptide, or a bispecific PD-L1×CD40-binding polypeptide, or a dimer thereof) may be administered to a subject in a therapeutically effective amount. According to the methods of the present disclosure, the polypeptide or protein described herein may be administered to a subject by a variety of modes of administration, including, for example, intramuscular, subcutaneous, intravenous, intraatrial, intraarticular, parenteral, intranasal, intrapulmonary, transdermal, intrapleural, intrathecal, and oral routes of administration. For prophylactic and therapeutic purposes, the antagonist may be administered to a subject in a single bolus delivery, over an extended period of time via continuous delivery (e.g., continuous transdermal delivery), or in a repeated administration protocol (e.g., hourly, daily, weekly, or monthly).

[0147] Thus, in some embodiments, the present disclosure provides a method of treating a subject, the method comprising administering a therapeutically effective dose of a pharmaceutical composition of the present disclosure to the subject. In some embodiments, the subject is afflicted with cancer. In some embodiments, the cancer expresses or overexpresses PD-L1. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a carcinoma. In some embodiments, the cancer is head and neck cancer, melanoma, lung cancer, brain cancer, thymus cancer, breast cancer, liver cancer, pancreatic cancer, kidney cancer, ovarian cancer, bladder cancer, gastrointestinal cancer, or colon cancer. In some embodiments, the patient is afflicted with head and neck squamous cell carcinoma, melanoma, or cancer of the lung, brain, thymus, breast, liver, pancreas, kidney, ovary, bladder, gastrointestinal tract, or colon.

[0148] In some embodiments, the present disclosure provides methods of treating or ameliorating symptoms of cancer (e.g., a cancer characterized by expression or overexpression of PD-L1) in a subject, the method comprising administering to the subject a pharmaceutical composition comprising a bispecific polypeptide of the present disclosure.

[0149] Provided herein are the binding polypeptides (e.g. bispecific PD-L1-binding polypeptides, bispecific CD40-binding polypeptides, or bispecific PD-L1×CD40-binding polypeptides), dimeric proteins, and compositions disclosed herein for use as medicaments.

[0150] Determination of effective dosages in this regard is typically based on animal model studies and subsequent human clinical trials, and is guided by determining effective dosages and administration protocols that significantly reduce the incidence or severity of the disorder of interest in the model subjects. The effective dose of the compositions of the present disclosure will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other drugs being administered, whether the treatment is prophylactic or therapeutic, and the specific activity of the composition itself and its ability to elicit the desired response in the individual. The patient will be a human, although depending on the disease, the patient may be a non-human mammal. Typically, dosing regimens will be adjusted to obtain the optimal therapeutic response, i.e., to optimize safety and efficacy. Accordingly, a therapeutically effective amount is also one in which the beneficial effects of administering a bispecific PD-L1-binding polypeptide, bispecific CD40-binding polypeptide, or bispecific PD-L1×CD40-binding polypeptide of the present disclosure, as described herein, outweigh any undesirable side effects.

[0151] In some embodiments, the dosage of the pharmaceutical composition may range from about 0.1 μg to about 100 mg / kg or about 1 μg / kg to about 50 mg / kg, more typically about 10 μg to about 5 mg / kg of the subject's body weight. In more specific embodiments, the effective amount of the agent is about 1 μg / kg to about 20 mg / kg, about 10 μg / kg to about 10 mg / kg, or about 0.1 mg / kg to about 5 mg / kg. In certain embodiments, the pharmaceutical composition is administered at a dose of about 100 μg / kg or less. In some embodiments, a pharmaceutical composition comprising a bispecific PD-L1-binding polypeptide, bispecific CD40-binding polypeptide, or bispecific PD-L1×CD40-binding polypeptide of the present disclosure is administered to a patient by intravenous injection at a dose of about 20 μg / kg, about 40 μg / kg, about 80 μg / kg, or about 200 μg / kg.

[0152] Dosage within this range can be achieved by single or multiple administrations, including, for example, multiple administrations per day or daily, weekly, biweekly or monthly administrations.For example, in certain variants, regimen consists of first administration followed by multiple subsequent administrations at weekly or biweekly intervals.Another regimen consists of first administration followed by multiple subsequent administrations at monthly or bimonthly intervals.Alternatively, administration can be irregular, as indicated by monitoring the clinical symptoms of disorder.

[0153] Dosages of pharmaceutical compositions containing the polypeptides or proteins described herein can be varied by the attending clinician to maintain the desired concentration at the target site. For example, if an intravenous delivery mode is selected, the local concentration of the drug in the bloodstream of the target tissue can range from about 0.01 to 50 nanomoles per liter of composition, and in some cases from about 1.0 nanomoles per liter to 10, 15, or 25 nanomoles per liter, depending on the subject's condition and the estimated measured response. Higher or lower concentrations can be selected based on the mode of delivery, e.g., transepidermal delivery versus delivery to a mucosal surface. Dosages may also need to be adjusted based on the release rate of the administered formulation, e.g., nasal spray versus powder, sustained-release oral or injectable particles, transdermal formulations, etc. To achieve the same serum concentration level, for example, sustained-release particles with a release rate of 5 nanomoles (under standard conditions) would be administered at a dose approximately twice that of particles with a release rate of 10 nanomoles.

[0154] The proteins and polypeptides described herein may also be administered at a daily dosage of about 0.001 to about 10 milligrams (mg) per kilogram (mpk) of body weight, preferably administered as a single dose or in divided doses about 2 to 6 times daily. For administration to adult human patients, therapeutically effective amounts may be administered in doses ranging from about 0.2 mg to about 800 mg per dose, including, but not limited to, 0.2 mg per dose, 0.5 mg per dose, 1 mg per dose, 5 mg per dose, 10 mg per dose, 25 mg per dose, 100 mg per dose, 200 mg per dose, and 400 mg per dose, with multiple, usually consecutive, daily doses being administered over the course of treatment. The proteins and polypeptides described herein may be administered at different times of day. In one embodiment, an optimal therapeutic dose may be administered in the evening. In another embodiment, an optimal therapeutic dose may be administered in the morning. Thus, the total daily dosage of the proteins and polypeptides described herein may, in one embodiment, range from about 1 mg to about 2 g, often from about 100 mg to about 1.5 g, and most often from about 200 mg to about 1200 mg. For a typical 70 kg adult human, the total daily dose of an anti-5T4 therapeutic agent may range from about 2 mg to about 1200 mg, and often, as noted above, from about 0.2 mg to about 800 mg.

[0155] Subjects for administration of the proteins of the present disclosure include patients at high risk of developing a particular cancer and patients with existing cancer. Typically, the subject has been diagnosed with the cancer for which treatment is sought. Furthermore, the subject can be monitored for any changes during the course of treatment. Also, in some variations, the subject does not suffer from another disease or disorder that requires treatment.

[0156] In preventive applications, pharmaceutical compositions or drugs containing the proteins of the present disclosure are administered to patients susceptible to or otherwise at risk of a particular disorder in an amount sufficient to eliminate or reduce the risk of the disorder or delay its onset. In therapeutic applications, pharmaceutical compositions or drugs containing the proteins of the present disclosure are administered to patients suspected of or already suffering from such a disorder in an amount sufficient to cure or at least partially arrest the symptoms of the disorder and its complications. An amount appropriate to achieve this is referred to as a therapeutically effective dose or amount. In both preventive and therapeutic regimens, agents are typically administered in several dosages until a sufficient response (e.g., inhibition of inappropriate angiogenic activity) is achieved. Responses are typically monitored, and repeat dosages are given if the desired response begins to wane.

[0157] To identify the target patient for treatment by the method of the present disclosure, acceptable screening methods can be used to determine the risk factors associated with specific disorders or to determine the status of the existing disorders identified in the subject.Such methods can include, for example, determining whether an individual has blood relatives diagnosed with a specific disorder.Screening methods can also include, for example, conventional workup to determine the familial status of a specific disorder known to have a genetic component.For example, various cancers are also known to have a specific genetic component. The hereditary component of cancer includes, for example, mutations in multiple transforming genes (e.g., Ras, Raf, EGFR, cMet, etc.), the presence or absence of certain HLA and killer inhibitory receptor (KIR) molecules, or mechanisms by which cells can directly or indirectly regulate the immunosuppression of cells such as NK cells and T cells (see, for example, Ljunggren and Malmberg, Nature Rev. Immunol. 7:329-339, 2007; Boyton and Altmann, Clin. Exp. Immunol. 149:1-8, 2007). For this purpose, nucleotide probes can be routinely used to identify individuals with genetic markers associated with a particular disorder of interest. Furthermore, a wide variety of immunological methods useful for identifying markers of specific disorders are known in the art. For example, various ELISA immunoassay methods using monoclonal antibody probes to detect antigens associated with specific tumors are available and well known in the art. Screening can be performed as indicated by known patient symptoms, age factors, associated risk factors, etc. These methods allow clinicians to routinely select patients in need of the methods described herein for treatment. According to these methods, targeting of inflammatory diseases or disorders described herein can be carried out as a stand-alone treatment program or as a follow-up, adjunctive, or coordinated treatment regimen to other treatments.

[0158] Pharmaceutical compositions comprising the proteins and polypeptides described herein (e.g., the bispecific PD-L1-binding polypeptides, bispecific CD40-binding polypeptides, or bispecific PD-L1×CD40-binding polypeptides of the present disclosure) may be supplied as kits comprising a container containing the pharmaceutical composition described herein. The pharmaceutical composition may be provided, for example, in the form of an injectable solution for single or multiple doses, or as a sterile powder to be reconstituted prior to injection. Alternatively, such kits may include a dry powder disperser, liquid aerosol generator, or nebulizer for administering the pharmaceutical composition. Such kits may further include written information regarding the indications and usage of the pharmaceutical composition.

[0159] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited herein, including, but not limited to, patents, patent applications, articles, books, and treatises, are expressly incorporated herein by reference in their entirety for all purposes. In the event that one or more of the incorporated documents or portions of documents defines a term that contradicts a term's definition in this application, the definition set forth in this application shall prevail. However, the mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be construed as, an admission or any suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0160] In describing the present invention, any concentration range, percentage range, ratio range, or integer range is understood to include any integer value within the recited range, and, where appropriate, fractions thereof (such as 1 / 10 and 1 / 100 of an integer), unless otherwise specified. As used herein, the terms "a" and "an" should be understood to refer to "one or more" of the recited components, unless otherwise specified. The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include" and "comprise" are used interchangeably.

[0161] The present disclosure will be further clarified by the following examples, which are intended to be purely illustrative of the disclosure and not limiting in any way. [Example]

[0162] Example 1. PD-L1-expressing CHO cells and production of recombinant extracellular domain protein Protein sequences defining the full-length and extracellular domain (ECD) of human, non-human primate, and mouse PD-L1 were obtained from the Genbank database and are listed in Table 4. The AFH protein sequence consists of an AviTag, a FLAGtag, and a His tag, representing a set of C-terminal tags for purification, detection, and biotin-based labeling. mFc represents the protein sequence of the mouse IgG2a hinge and Fc region. PD-L1 hu-IgV mu-IgC ECD is a hybrid of human and mouse PD-L1 ECD. Table 4. SEQ ID NOs of cell lines and PD-L1 constructs for recombinant protein production [Table 3]

[0163] DNA containing the nucleotide sequences encoding the proteins listed in Table 4 was synthesized at IDT, Coralville, IA, and inserted into either expression vectors suitable for mammalian cell expression and secretion, or expression vectors suitable for cell surface expression, including the ability to generate stable transfectants under selective pressure. These reagents were used to evaluate the cross-reactivity and binding strength of the anti-PD-L1 binding domains to human PD-L1 and species used in potential toxicity assessments. Human embryonic kidney fibroblast (HEK)-293 cells grown in suspension culture were transiently transfected with a DNA expression vector encoding the AFH tag. After several days of culture, the conditioned medium was clarified by centrifugation and sterile filtration. Protein purification was performed using immobilized metal affinity chromatography (IMAC) coupled with size-exclusion chromatography (SEC). SEC removed aggregation and clipping products as well as other host cell contaminants. SEC was also used to buffer-exchange the protein into phosphate-buffered saline (PBS). Final purity was determined by analytical SEC and typically exceeded 90%. Protein batches were sterile filtered and stored at 4°C for use within the next week or frozen in aliquots in a -80°C freezer.

[0164] Plasmid DNA encoding the full-length construct was digested with restriction enzymes, precipitated with ethanol, and then dissolved in ultrapure water followed by Maxcyte Electroporation Buffer. The linearized DNA was transfected into Chinese hamster ovary (CHO)-K1SV cells (CDACF-CHO-K1SV cells (ID code 269-W3), Lonza Biologics) by electroporation. The transfected cells were transferred from the electroporation cuvette to a T75 culture flask, allowed to rest, and then gently resuspended in 15 mL of CD CHO medium supplemented with 6 mM L-glutamine in a T150 flask. The flask was placed in a 37°C, 5% CO2 incubator and allowed to recover for 24 hours before being placed under selection conditions. The day after transfection, the cells were centrifuged at 1000 RPM for 5 minutes and resuspended in CD CHO medium containing 1X GS supplement and 50 μM MSX. After recovering the bulk population from the initial selection, cells were assessed for surface expression using commercially available reagents, and representative vials were frozen. To obtain clones with various expression levels, cells were sorted by flow cytometry, plated by limiting dilution, and expanded for 2 weeks. Wells were imaged with a Clone Select Imager during incubation to identify growth-positive wells. Only wells with good image quality were selected for further expansion and characterization of surface expression by flow cytometry. All clones were frozen in a bank, up to 30 vials per clone.

[0165] Example 2. General expression and purification of PD-L1 and CD40 binding molecules and antibodies The monospecific and bispecific PD-L1 and CD40 binding molecules disclosed herein were produced by transient transfection of either HEK293 or CHO cells. Cultures were cleared of cells, cell debris, and insoluble material by centrifugation and / or filtration. Recombinant homodimeric proteins were captured from the clarified conditioned medium using Protein A affinity chromatography (ProA). Preparative size-exclusion chromatography (Prep SEC) was routinely performed to further purify the protein, homogenize it, and buffer-exchange it into PBS. After each of the ProA and Prep SEC purification steps, protein purity was verified by analytical size-exclusion chromatography (Analytical SEC) on an Agilent HPLC.

[0166] Heteromeric proteins, in which two or more peptide chains assemble to form soluble protein complexes, were expressed using CHO cells transiently transfected with separate plasmids for each peptide chain. In some cases, the plasmids were transfected at equal ratios. If one peptide chain was observed to express significantly better than the other(s), the plasmid ratio was changed to transfect more of the lower-expressing plasmid. ProA was used to capture proteins from cell culture supernatants using wash steps and a low-pH elution step. Preparative SEC was used to remove aggregated proteins and exchange the sample into PBS. In some cases, a second ProA chromatography step was performed. After washing the column with PBS, the protein was eluted using a decreasing pH gradient (neutral to acidic). In some cases, cation exchange chromatography was used to further purify the heterodimers to remove low-MW, homodimer, and unpaired peptide chain contaminants.

[0167] In most cases, the final protein batch was buffer exchanged into PBS as part of the SEC purification process, adjusted to 1 mg / mL, sterile filtered, and stored at 4°C until needed or otherwise specified. Protein concentrations were determined from absorbance at 280 nm using the theoretical extinction coefficient calculated from the amino acid sequence.

[0168] Endotoxin levels were measured using an Endosafe PTS instrument according to the manufacturer's instructions. This ensured that the results of the in vitro activity assays were not confounded by the presence of endotoxin. Analytical SEC, along with peak area integration, was used to quantify sample purity. In some cases, the resolution of analytical SEC was insufficient to separate the desired heterodimer product from product-related contaminants. Capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) was used as a secondary method to assess product purity. Reducing and nonreducing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gels were run with molecular weight (MW) standards to confirm product purity and estimate MW.

[0169] Example 3. Generation of PD-L1 antibodies by immunization of wild-type mice PD-L1-specific antibodies were isolated from a hybridoma library generated after immunization of BALB / c mice with the recombinant hybrid human / mouse antigen PDL01017 at Precision Antibody, Columbia, MD. Supernatants of hybridoma clones were assayed by ELISA, and identified wells were confirmed for specific binding using flow cytometry against CHO cells transfected with human and cynomolgus PD-L1 (PDL01001 and 03). Positive clones were selected for expansion, and viable cells were frozen for RNA extraction and variable domain analysis. Supernatants were stored for further analysis.

[0170] The variable heavy (VH) and light (VL) domain sequences of selected hybridoma clones were obtained by RT-PCR after total RNA isolation. Briefly, total RNA was isolated from the hybridoma clone cell bank using Qiagen's RNeasy Plus Kit (Qiagen, Venlo, Netherlands). 400 ng of total RNA was used for first-strand cDNA synthesis using oligo-dT and Superscript IV (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's protocol. After cDNA synthesis, variable region cDNA was amplified using 1 μL of cDNA and a set of primer mixes for mouse IgG VH, Vκ, and Vλ (Novagen Mouse Ig-Primer Set, EMD Millipore, Temecula, CA). The PCR products of each clone were directly sequenced using reverse (constant domain) PCR primers and standard Sanger sequencing. DNA sequence analysis identified 10 distinct antibodies: 2C11, 3G5, 3G11, 5F9, 5F11, 6F6, 7A3-1, 7A3-2, 7H11, and 10B8. The sequences were then converted into scFv format in VH-VL and VH-VL orientations with 4xG4S linkers by amplifying the variable domains using specific primers containing overlapping sequences (see Table 5 for a list of constructs), and assembled into mammalian expression vectors as scFv-Fc with human IgG1 WT Fc using the NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs, Beverly MA). The positive control anti-PD-L1 antibodies 5F11 and 7A3 were constructed as chimeric antibodies. See PDL01050 and PDL01051. Table 5. SEQ ID NOs of anti-PD-L1 hybridoma-derived scFv-Fc constructs and positive controls [Table 4]

[0171] Example 4. Humanization of PD-L1-specific clone 5F9 in scFv format After evaluation of hybridoma-derived antibodies (described in Examples 6, 7, and 8), clone 5F9 was selected for full humanization and optimization. The primary goal of humanization was to eliminate as much mouse-derived sequence as possible to minimize potential immunogenicity while optimizing the binding and stability characteristics of the binding domain. Clone 5F9 anti-PD-L1 mouse monoclonal antibody (PDL01034, VH SEQ ID NO: 276, VL SEQ ID NO: 277) in scFv-hFc format was humanized through multiple rounds of CDR-grafting and purification using the BioLuminate software package (Schrodinger, LLC, New York, USA). A homology model of mouse clone 5F9 was created based on PDB IDs 5B3J, 5VYF, and 6HHC, and the most geometrically suitable and homologous human framework for CDR-grafting was identified using the software's default and modified settings. Seven CDR-grafted molecules were produced on a small scale and tested for binding to cells expressing full-length human or cynomolgus PD-L1 without purification. After scale-up and purification, their binding kinetics to recombinant human PD-L1 antigen were measured using a Biacore T200 (GE Healthcare Life Sciences, USA) and T using an Uncle instrument (Unchained Labs, USA). m and T aggStability, as measured by β-glucan, as well as expression and initial aggregation levels, were recorded (data not shown). In stage 2, molecules PDL01060 and PDL01065 were further germlined, and molecule PDL01085 was shown to have similar binding properties to the parent mAb 5F9 and good thermal stability (data not shown). In stage 3, additional framework residues were mutated in sets and combinations to convert the murine residues of PDL01085 to human germline sequences IGHV3-48*01 and IGHJ4*01 for the heavy chain and IGKV1-39*01 and IGKJ2*01 for the light chain. Molecule PDL01152 was identified as having the best combination of binding, function, and developability properties. All non-germlined residues in the framework are essential for either binding or stability. The progression of humanization from murine to humanized sequence is shown in the amino acid alignment in Figure 1 (from murine anti-PD-L1 clone 5F9 (PDL01034) to humanized PDL01152).

[0172] All antibody protein engineering was performed at the protein sequence level. Genes corresponding to the designed proteins were synthesized by Integrated DNA Technologies Inc., Coralville, IA, USA, using an online gene design tool for optimized expression in mammalian systems. The synthetic genes were combined with each other or with expression vectors using the NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs, Beverly, MA) or standard molecular biology techniques and methods generally disclosed in, for example, PCT Application Publication No. WO2007 / 146968, U.S. Patent Application Publication No. 2006 / 0051844, PCT Application Publication No. WO2010 / 040105, PCT Application Publication No. WO2010 / 003108, and U.S. Patent No. 7,166,707. DNA sequences were verified using Sanger sequencing at GENEWIZ, South Plainfield, NJ, USA.

[0173] Example 5. Production and biophysical characterization of anti-PD-L1 scFv variants Different humanized versions of clone 5F9 scFv were produced as monospecific DNA constructs by attaching the scFv sequence to the N-terminus of the wild-type IgG1 Fc region. After transient expression and purification of sufficient quantities of the constructs, these constructs were characterized for expression, thermal stability by differential scanning fluorimetry (DSF), and binding affinity to human PD-L1 ECD by SPR (as described in Example 6).

[0174] The midpoint of the first melting transition (T m1 ) was measured using DSF. m1 was used to reflect the temperature required to unfold the first, or least stable, binding domain in the construct. DSF was performed using an Uncle instrument from Unchained Laboratories. Samples were analyzed at 1 mg / mL in PBS using intrinsic fluorescence (no additional dye was used to assess protein unfolding). The parent construct, PDL01034, had the lowest T recorded. m1 was 55.1°C, while the derivative constructs all had values ​​above 63°C, indicating that they were all more thermostable.

[0175] As shown in Table 6, testing of binding to human PD-L1 ECD by SPR revealed various effects on affinity during humanization. Two variants (PDL01085 and PDL01152) retained similar binding affinity to the parent (KD of approximately 1 nM), while two (PDL01060 and PDL01065) exhibited reduced affinity and higher KDs (217 and 16 nM, respectively). Furthermore, significant increases in expression were obtained with three variants (PDL01060, PDL01085, and PDL01152) compared to the unmodified parent construct (PDL01034). Expression and T m1 The improved values ​​suggest that the variants maintain similar binding affinity to the parent binding domain while exhibiting improved stability and solubility, which are considered beneficial properties for therapeutic protein drugs. Table 6. Summary of expression, thermostability, and affinity of preferred anti-PD-L1 variants compared to the parental sequence [Table 5]

[0176] Example 6. Methodology for determining the binding affinity of anti-PD-L1 binding domains using surface plasmon resonance (SPR) SPR binding affinity studies of monospecific and bispecific proteins binding to the recombinant human PD-L1 ectodomain (ECD) were performed on a Biacore 8K or Biacore T200 system in HBS-EP+ buffer (Cytiva, BR100826) containing 0.1% bovine serum albumin (BSA) at 25°C. Mouse anti-human IgG (Cytiva, BR100839) at 25 μg / ml in 10 mM sodium acetate, pH 5.0, was immobilized to each flow cell of a CM5 research-grade sensor chip (Cytiva, BR100530) using standard amine coupling chemistry at a density of 2,000–4,000 response units (RU). Approximately 40 nM of each anti-PD-L1 protein in HBS-EP+ containing 0.1% BSA buffer was captured onto the flow cell along with immobilized anti-human IgG at a flow rate of 10 μL / min for up to 30 seconds, leaving one unmodified flow cell surface as a reference. Using multi-cycle kinetics mode, buffer blank and five different concentrations of ECD ranging from 0.5 nM to 243 nM were injected sequentially onto each flow cell at 30 μL / min, with association times varying from 300 to 600 seconds and dissociation times varying from 600 to 1200 seconds. Regeneration was achieved by injecting 3 M MgCl2 at a flow rate of 30 μL / min for up to 40 seconds, followed by 1 minute of stabilization in HBS-EP+ containing 0.1% BSA buffer.

[0177] The sensorgrams obtained from kinetic SPR measurements were analyzed using the double subtraction method. The signal from the reference flow cell was subtracted from the analyte binding response obtained from the flow cell with captured ligand. The buffer blank response was then subtracted from the analyte binding response, and the final double-referenced data was analyzed using Biacore T200 evaluation software (v2.0, Cytiva). The data were globally fitted to derive kinetic parameters. All sensorgrams were fitted using a simple one-to-one binding model.

[0178] Several monospecific anti-PD-L1 scFv-Fc proteins were assessed for their binding affinity to monomeric PD-L1 ECD using SPR (see Table 6).

[0179] Example 7: Binding of PD-L1 binding molecules to human and cynomolgus PD-L1 expressing CHO cell lines The PD-L1 scFv binding domain was evaluated using the MesoScale Discovery (MSD) assay. 50,000 cells (CHOK1SV / huPDL1, CHOK1SV / cynoPDL1, and parental CHOK1SV) were plated into a multi-array 96-well high-binding plate (MSD) and then blocked with 40% FBS / PBS at 37°C for 1 hour. The plate was washed, and titrated test samples were added and incubated at room temperature for 1 hour. The plate was washed again, and 0.5 mg / ml goat anti-human sulfo-TAG detection antibody (MSD) was added and incubated at room temperature for 1 hour. The plate was washed, and 1X detergent-free MSD Read Buffer was added, and the plate was read on a Meso QuickPlex SQ120 plate reader using Discovery Workbench software (MSD).

[0180] As shown in Figures 2A-C, all PD-L1 antibodies bound to both human (Figure 2A) and cynomolgus monkey (Figure 2B) PD-L1-expressing cells. Only one antibody, PDL01049, exhibited increased nonspecific binding to parental CHOK1SV cells (Figure 2C) and was excluded from further study.

[0181] After further evaluation, 5F9 was determined to be the desired PD-L1 binding domain and underwent humanization. Figures 2d-2F show two PD-L1 leads, PDL01060 and PDL01065, binding to human (Figure 2d), cynomolgus monkey (Figure 2E), and parental CHOK1SV (Figure 2F) cells using the MSD platform assay. Both humanized molecules bound well to PD-L1 and showed no nonspecific binding to the parental cells.

[0182] Example 8: Binding of PD-L1 binding molecules to various human PD-L1-expressing cell lines Cell-binding studies were completed to demonstrate that the PD-L1 scFv binding domain bound well to cells expressing PD-L1 (CHOK1SV / huPD-L1, CHOK1SV / cynoPD-L1, K562, and KG-1), but not to cells lacking PD-L1 expression (Jurkat, U-937, C4-2B, or parental CHOK1SV). Binding studies were performed using the highly sensitive MSD assay platform.

[0183] Figures 3A-3B graphically represent the binding of 5F9 clone PDL01034 (Figure 3A) and 6F6 clone PDL01036 (Figure 3B) to these various cell lines. As expected, both molecules strongly bound to human and cynomolgus monkey PD-L1-expressing CHOK1SV and KG-1, both of which express high levels of PD-L1. There was additional weak binding to K562, which expresses low levels of PD-L1. Importantly, neither molecule bound to the negative control cell line.

[0184] Example 9: CD40-expressing CHO cells and production of recombinant extracellular domain protein Protein sequences defining the full-length and extracellular domains (ECDs) of human and non-human primates CD40 were obtained from the Genbank database and are listed in Table 7. The HAC protein sequence consists of a His-tag, an AviTag, and a C-tag, representing a set of C-terminal tags for purification, detection, and biotin-based labeling. mFc represents the protein sequence of the mouse IgG2a hinge and Fc region. Table 7. SEQ ID NOs of CD40 constructs for cell lines and recombinant protein production [Table 6]

[0185] DNA containing the nucleotide sequences of the proteins listed in Table 7 was synthesized by Integrated DNA Technologies Inc., Coralville, IA, USA, and inserted into either an expression vector suitable for mammalian cell expression and secretion, or an expression vector suitable for cell surface expression, including the ability to generate stable transfectants under selective pressure. These reagents were used to evaluate the cross-reactivity and binding strength of the anti-CD40 binding domain to human CD40 and species used in potential toxicity assessments. Human embryonic kidney fibroblast (HEK)-293 cells grown in suspension culture were transiently transfected with a DNA expression vector encoding the HAC tag. After several days of culture, the conditioned medium was clarified by centrifugation and sterile filtration. Protein purification was performed using immobilized metal affinity chromatography (IMAC) coupled with size-exclusion chromatography (SEC). SEC removed aggregation and clipping products as well as other host cell contaminants. SEC was also used to buffer-exchange the protein into phosphate-buffered saline (PBS). Final purity was determined by analytical SEC and typically exceeded 90%. Protein batches were sterile filtered and stored at 4°C for use within the next week or frozen in aliquots in a -80°C freezer.

[0186] Plasmid DNA encoding the full-length construct was digested with restriction enzymes, precipitated with ethanol, and then dissolved in ultrapure water followed by Maxcyte Electroporation Buffer. The linearized DNA was transfected into CHO-K1SV cells (CDACF-CHO-K1SV cells (ID code 269-W3), Lonza Biologics) by electroporation. The transfected cells were transferred from the electroporation cuvette to a T75 culture flask, allowed to rest, and then gently resuspended in 15 mL of CD CHO medium supplemented with 6 mM L-glutamine in a T150 flask. The flask was placed in a 37°C, 5% CO2 incubator and allowed to recover for 24 hours before being placed under selection conditions. The day after transfection, the cells were centrifuged at 1000 RPM for 5 minutes and resuspended in CD CHO medium containing 1X GS supplement and 50 μM MSX. After recovering the bulk population from the initial selection, cells were assessed for surface expression using commercially available reagents, and representative vials were frozen. To obtain clones with various expression levels, cells were sorted by flow cytometry, plated by limiting dilution, and expanded for 2 weeks. Wells were imaged with a Clone Select Imager during incubation to identify growth-positive wells. Only wells with good image quality were selected for further expansion and characterization of surface expression by flow cytometry. All clones were frozen in a bank, up to 30 vials per clone.

[0187] Example 10. Generation of CD40 antibodies by immunization of wild-type mice CD40-specific antibodies were isolated from a hybridoma library generated after immunization of BALB / c mice with recombinant human antigen CD4001005 at Precision Antibody, Columbia, MD. Supernatants of hybridoma clones were assayed by ELISA, and identified wells were confirmed for specific binding using flow cytometry on CHO cells transfected with human and cynomolgus CD40. Positive clones were selected for expansion, and viable cells were frozen for RNA extraction and variable domain analysis. Supernatants were stored for further analysis.

[0188] The variable heavy (VH) and light (VL) domain sequences of selected hybridoma clones were obtained by RT-PCR after total RNA isolation. Briefly, total RNA was isolated from the hybridoma clone cell bank using Qiagen's RNeasy Plus Kit (Qiagen, Venlo, Netherlands). 400 ng of total RNA was used for first-strand cDNA synthesis using oligo-dT and Superscript IV (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's protocol. After cDNA synthesis, variable region cDNA was amplified using 1 μL of cDNA and a set of primer mixes for mouse IgG VH, Vκ, and Vλ (Novagen Mouse Ig-Primer Set, EMD Millipore, Temecula, CA). The PCR products of each clone were directly sequenced using reverse (constant domain) PCR primers and standard Sanger sequencing. DNA sequence analysis identified 16 distinct antibodies: 1A7, 1B12, 1D5, 4G10, 5B9, 5E3, 6A4, 6B3, 7A8, 7G9, 7H12, (CD4001014-24), 1G4, 2H7, 6B12, 9A6, and 9F1 (CD4001036, 38, 40, 42, 44) (see Table 2). The sequences were then converted into scFvs of both VH-VL and VL-VH orientations by amplifying the variable domains using specific primers containing overlapping sequences. The scFv-encoding fragments for the anti-CD40 binding domain were fused to the N-terminus of human WT IgG1 Fc, and the anti-PDL1 scFv 5F9 was fused to the C-terminus. These constructs were assembled into mammalian expression vectors using the NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs, Beverly, MA). Table 8. SEQ ID NOs of anti-CD40 hybridoma-derived scFv-Fc constructs [Table 7]

[0189] Example 11. CD40 ECD domain mapping of hybridoma clones Taking advantage of the lack of binding activity of hybridoma anti-CD40 clones to the modular domain structure of mouse CD40 and CD40 ECD, we designed a set of human / mouse domain-swap hybrid constructs. Human CD40 ECD was divided into three domains at the junctions at His78 and His122. These constructs were expressed as full-length hybrid CD40 proteins and transiently expressed on the surface of CHO cells. An N-terminal 2xFLAG tag was used to determine the expression levels of all constructs and to normalize binding signals. The transmembrane and intracellular domains were derived from human CD40 for all constructs. Table 9 lists the constructs used in domain mapping. Table 9. SEQ ID NOs of CD40 ECD domain swap constructs for domain mapping of hybridoma clones [Table 8]

[0190] Six hybridoma clones in scFv format, 1A7, 2H7, 5B9, 1D5, 1G4, and 9A7, were tested by flow cytometry for binding to human / mouse hybrid CD40 ECD expressed in CHO cells. The anti-FLAG antibody strongly stained all cells bearing the hybrid construct, as shown in Table 10. Table 10. Relative expression of hybrid CD40 on the surface of CHO cells [Table 9]

[0191] The signals from the hybridoma clones were normalized to both the FLAG signal and the huCD40 signal and are shown in Table 11. The data show that 1A7 binds to domain 2, 2H7 binds primarily to domains 2 and 1, 5B9 binds to domain 1, and 1D5, 1G4, and 9A7 hybridoma clones bind to domain 3. Table 11. Relative % binding of hybridoma clones to human / mouse hybrid CD40 on the surface of CHO cells [Table 10]

[0192] Example 12. Blocking CD40 / CD40l interactions by hybridoma clones in scFv format Two sets of experiments were performed to examine the ability of hybridoma antibody clones 1D5, 2H7, and 5B9 (constructs CD4001066, 64, and 65) to block CD40 receptor-CD40 ligand binding on cells. In the first experiment, CD40-expressing CHO cells were saturated with CD40L followed by antibody binding; in the second experiment, CD40-expressing CHO cells were saturated with antibody followed by CD40L. Both experiments demonstrate that 1D5 and 2H7 block the CD40 / CD40L interaction, whereas 5B9 does not. All three hybridoma clones were in the ADAPTIR format, containing an anti-PD-L1 scFv at the N-terminus and an anti-CD40 scFv at the C-terminus.

[0193] For both assays, 100,000 CD40-expressing HEK293 cells were plated. In the first assay, a dilution of His-tagged CD40L (BPS Bioscience) was added to the cells and incubated on ice for 1 hour. The cells were washed, and 100 nM anti-CD40 antibody was added, incubated on ice for 30 minutes, and then washed repeatedly. The cells were then incubated with PE-labeled goat anti-huIgG F(ab')2 (Jackson). After a 30-minute incubation, the cells were washed and analyzed by flow cytometry.

[0194] In the second assay, serially diluted anti-CD40 antibodies were added and incubated on ice for 1 hour. Cells were washed, and 100 nM CD40L was added for 30 minutes. After repeated washing, PE-labeled anti-His (Biolegend) was added. After a 30-minute incubation, cells were washed and analyzed by flow cytometry. All incubations and washes were performed in staining buffer. All samples were collected using a BD™ LSR-II Flow and analyzed with FlowJo flow cytometry analysis software. The mean fluorescence intensity (MFI) of bound molecules on cells was determined after exclusion of doublets.

[0195] Figure 4A shows the percentage of anti-CD40 antibodies blocked by preincubation with CD40L. Both CD4001064 (2H7) and CD4001066 (1D5) are blocked by CD40L. In contrast, CD4001065 (5B9) does not significantly reduce binding to CD40 when competing with CD40L binding. Alternatively, Figure 4B shows the MFI of CD40L bound to CD40-expressing cells after preincubation with anti-CD40 antibodies. Here, CD4001064 (2H7) and CD4001066 (1D5) block CD40L binding starting at 10 nM.

[0196] Example 13. Anti-PD-L1 and anti-CD40 bispecific proteins with different structures and valencies The optimal distance and geometry for forming an immune synapse between PD-L1-expressing tumor cells and CD40 receptor-expressing cells is unknown. Although the epitopes of our anti-PD-L1-specific and anti-CD40-specific binding domains are predetermined and cannot be altered, we tested different bispecific structures to achieve optimal functional activity. Valency was also investigated by producing and testing molecules with heterodimeric structures in the ADAPTIR-FLEX format, containing one or two binding domains for PD-L1 and CD40, located at either the N- or C-terminus of the Fc region (see Table 12). In addition, the effect of altering the order of the scFv domains (VH-VL vs. VL-VH) was investigated. In addition to affecting the binding affinity and functional performance of ADAPTIR and ADAPTIR-FLEX, these structural changes may also have a significant impact on the expression levels and stability of the bispecific proteins, and were investigated as part of this study. Table 12. SEQ ID NOs and descriptions of bispecific constructs with different structures and valencies [Table 11] JPEG2026501724000023.jpg28159

[0197] To assess the impact of binding domain, shape, and valency, molecular combinations were first tested for binding to both CD40-expressing CHOK1SV cells and PD-L1-expressing CHOK1SV cells via flow cytometry (Figures 5A and 5B, respectively). The experimental methods and analyses were similar to those described above. During this matrix evaluation, several overall observations were determined. First, CD40 binding was reduced when the anti-CD40 binding domain was at the C-terminus, regardless of whether it was a monovalent or bivalent binding domain. Overall, monovalent CD40 binding was significantly lower than bivalent binding. In general, monovalent PD-L1 combined with bivalent CD40 and bivalent PD-L1 at the N-terminus exhibited higher PD-L1 binding than other combinations.

[0198] To quantify PD-L1 / PD-1 blocking activity, a human PD-1 NFAT luciferase reporter Jurkat line (BPS) was incubated with TCR activator PD-L1-expressing CHO target cells (BPS). 35,000 target cells were plated overnight in a 96-well assay plate. The following day, diluted bispecific samples were added and incubated for 30 minutes. 80,000 reporter cells were then added and incubated at 37°C for 6 hours. The plate was then equilibrated to room temperature, and 100 ml of RT Bio-Glo reagent (Promega) was added and incubated for 15 minutes. Luminescence was read using a MicroBeta 2450 Microplate Counter (Perkin Elmer). In the PD-L1 / PD-1 blocking assay shown in Figure 6, bivalent PD-L1 molecules were significantly better at blocking PD-L1 / PD-1, as measured by light generation, compared to molecules containing monovalent PD-L1. This effect was independent of the orientation of either binding domain or the valency of the anti-CD40 binding domain.

[0199] To examine CD40 activity, human CD40 / NFκB HEK293 luciferase reporter cells (BPs) were incubated with PD-L1-expressing target cells. 30,000 reporter cells were plated overnight in a 96-well assay plate. The following day, diluted bispecific samples and 50,000 target cells were added and incubated at 37°C for 6 hours. The plate was then equilibrated to room temperature, and 100 ml of RT Bio-Glo Reagent (Promega) was added and incubated for 10 minutes. Using the CD40 reporter assay shown in Figure 7A, it was determined that bivalent CD40 molecules induced higher downstream signaling than monovalent CD40 molecules. Furthermore, in the monovalent case, binding was further reduced when the C-terminus was present. Overall, the 5B9 binding domain produced the highest activity, followed by 1D5. The 2H7 binding domain generally induced the lowest CD40 activity. Using parental CHOK1SV cells (Fig. 7B), when PD-L1-mediated cross-linking was eliminated, higher levels of CD40 activity were observed with the 2H7 and 5B9 bivalent binding domains, which was more pronounced at the N-terminus.

[0200] Example 14. Humanization of CD40-specific clone 1D5 in scFv format After evaluation of hybridoma-derived antibodies, clone 1D5 was selected for full humanization and optimization. The primary goal of humanization was to eliminate as much murine-derived sequence as possible to minimize potential immunogenicity while optimizing the binding and stability characteristics of the binding domain. Humanization of the clone 1D5 anti-CD40 mouse monoclonal antibody was performed in scFv-hFc format with multiple rounds of CDR-grafting and purification using the BioLuminate software package (Schrodinger, LLC, New York, USA). A homology model of mouse clone 1D5 was created based on PDB ID 5KVD, and the most geometrically suitable and homologous human frameworks for CDR-grafting were identified using the software's default and modified settings. These were 6U6U, 5N7W, 6OKM, 1T3F, 1CE1, 2WUB, and 3NFS. Twenty-eight CDR-grafted molecules were produced on a small scale and tested for binding to cells expressing full-length human or cynomolgus CD40 without purification. Clones with parent-like binding (CD4001101-03) were purified at scale and their binding affinity determined by on-cell binding, T, using the Unchained apparatus (Unchained Labs, USA). m and T agg Stability by measuring ribosomal expression, as well as expression and initial aggregation levels, were recorded. In stage 2, the molecule CD4001103 was further germlined, and the humanized variant CD4041134-38 was similarly tested. Several additional mutation sets were tested in CD4001152-64, but none of the variants retained their binding activity in the Fc-scFv format. The humanization progress is depicted in Figure 8A for the VH chain and Figure 8B for the VL chain.

[0201] De novo humanization of the clone 1D5 anti-CD40 mouse monoclonal antibody was performed in hFc-scFv format with multiple rounds of CDR grafting and purification using the BioLuminate software package (Schrodinger, LLC, New York, USA). A homology model of mouse clone 1D5 was created based on PDB IDs 5KVD, 4M61, 4M7K, 5HDQ, and 4BZ1, and the most geometrically suitable and homologous human framework for CDR grafting was identified using default and modified settings of the software. Thirty-one CDR-grafted molecules were produced on a small scale and tested without purification for binding to cells expressing full-length human or cynomolgus monkey CD40. Ten clones with binding-matched to the parent mouse 1D5 were scaled up and purified to determine their binding affinity by on-cell binding and T cell binding using the Unchained Labs (Unchained Labs, USA). m and T agg The stability, as well as expression and initial aggregation levels, were recorded by measuring the expression level (CD4001171-1180). In stage 2, the molecule CD4001180 was further germlined and the humanized variant CD4041181-1207 was similarly tested. The final humanized sequence of the 1D5 antibody has a VL of CD4001193 and a VH of CD4001206. The humanization progress is depicted in Figure 8C for the VH chain and in Figure 8D for the VL chain.

[0202] Example 15. Production and biophysical evaluation of a partially humanized version of anti-CD40 clone 1D5 Different humanized versions of clone 1D5 scFv were produced as monospecific DNA constructs by linking the scFv sequence to the C-terminus of the wild-type IgG1 Fc region. After transient expression and purification of sufficient quantities of the constructs, these constructs were characterized for expression and thermostability by differential scanning fluorimetry (DSF).

[0203] The midpoint of the first melting transition (T m1 ) was measured using DSF. m1was used to reflect the temperature required to unfold the first, or least stable, binding domain in the construct. DSF was performed using an Uncle instrument from Unchained Laboratories. Samples were analyzed at 1 mg / mL in PBS using intrinsic fluorescence (no additional dye was used to assess protein unfolding).

[0204] These data showed that expression and thermostability were sometimes adversely affected by the elimination of mouse sequences (see Table 13). Many constructs had T > 60°C. m1 Some constructs showed high thermostability, but others were significantly reduced and excluded from further development. Expression was similarly variable, with measured titers ranging from 44.0 to a maximum of 360.6 μg / mL. Binding affinity was determined by on-cell binding (Example 21). These data, along with the extent of human sequence integration as described in Example 15, were used to explore general trends. Table 13. Summary of expression and thermostability of partially humanized anti-CD40 variants of clone 1D5 [Table 12]

[0205] Example 16. PTM removal of CD40-specific clone 1D5 by phage display The HCDR3 of the 1D5 anti-CD40 binding domain contains the DDG sequence, which contains two iso-aspartic acid isomerization motifs, DD and DG. To identify PTM-neutralizing variants, a phage display library was designed to mutate the DDG sequence to all 20 possible amino acids. The library was synthesized at IDT, Coralville, IA, and assembled into a phage display vector using the NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs, Beverly, MA). Standard phage display library generation and panning techniques were used. (Antibody Phage Display: Methods and Protocols, ed. Robert Aitken, 2009 Methods in Molecular Biology, vol. 562) For panning, we used site-specific biotinylated human CD40 ECD (CD4001006), streptavidin-coated magnetic beads (Dynabeads M-280 Streptavidin, Thermo Fisher Scientific), and a KingFisher mL Purification System (Thermo Fisher Scientific).

[0206] After the first round of panning with 200 nM biotin-CD40, there was no enrichment. A second round of panning with 10 nM biotin-CD40 resulted in a 10-fold enrichment over background, as did parallel panning without biotin-CD40. Round 3 panning used 2 nM biotin-CD40, resulting in a 10-fold enrichment over background. Each subsequent round of panning was performed with longer wash conditions: 10, 30, and 120 seconds, respectively. The phage output from round 3 was analyzed by colony PCR and sequencing of 96 clones. The results are shown in Table 14. The DDG high-risk PTM motif was mutated to several other sequences that likely retained binding to CD40. Table 14. Sequence enrichment analysis from the 1D5 HCDR3 DDG library [Table 13]

[0207] Example 17. Evaluation of the stability and manufacturability consequences of CD40-specific clone 1D5 PTM removal Following phage display (Example 16), variants of the DDG sequence that did not contain PTM risk were introduced into the parent PC401003 molecule. These constructs were then transiently expressed in CHO cells and purified as described above to examine the impact of incorporating alternative sequences into the anti-CD40 HCDR3. These comparisons included evaluation of expression, thermostability by DSF (described above), and binding affinity to human CD40 ECD by SPR.

[0208] Comparison of the constructs shows that transient CHO expression levels remain largely unchanged when the DDG sequence is altered. However, two constructs (DKG and DWG) that showed moderately reduced expression levels failed during purification. Further data are not reported, as indicated by a "-" in Table 15. The parent (PC401003 DDG) and the remaining three variants (TDH, DAG, and DQG) were successfully purified and further analyzed.

[0209] The thermal stability of these four constructs was assessed by measuring Tm1 using DSF as previously described. Compared to the parent, all PTM variants affected the thermal stability, resulting in a T m1 The binding affinity of the original PC401003 and the three PTM variants to human CD40 was further determined. Only TDH was significantly affected by the sequence changes, with its affinity to CD40 nearly 8-fold lower than that of PC401003. Table 15. Summary of expression, thermostability, and CD40 affinity of PDL1 x CD40 bispecific proteins using PTM-deleted variants of the CD40-binding domain [Table 14]

[0210] Example 18. Humanization of CD40-specific clone 5B9 in scFv format After evaluation of hybridoma-derived antibodies, clone 5B9 was selected for full humanization and optimization. The primary goal of humanization was to eliminate as much murine-derived sequence as possible to minimize potential immunogenicity while optimizing the binding and stability characteristics of the binding domain. Humanization of the clone 5B9 anti-CD40 mouse monoclonal antibody was performed in scFv-Fc format with multiple rounds of CDR-grafting and purification using the BioLuminate software package (Schrodinger, LLC, New York, USA). A homology model of mouse clone 5B9 was created based on PDB IDs 1KFA, 5Y9F, 2DQU, and 2A77, and the most geometrically suitable and homologous human framework for CDR-grafting was identified using the software's default and modified settings. Forty-nine CDR-grafted molecules were produced on a small scale and tested for binding to cells expressing full-length human or cynomolgus CD40 without purification. Fifteen clones (clone numbers CD4001086-1100) that matched the binding of the parental murine 5B9 were scaled up and purified, and their binding kinetics to recombinant human CD40 antigen were measured using a Biacore T200 (GE Healthcare Life Sciences, USA) or cell binding, stability by measuring Tm and Tag using an Uncle instrument (Unchained Labs, USA), and expression and initial aggregation levels were recorded. In Stage 2, the molecule CD4001100 was further germlined to the complete human sequence, generating 30 mutation combinations. These were initially screened from small-scale production runs, and the best two clones were scaled up and characterized. These were labeled clones CD4001132 and CD4001133. The progress of the VH chain humanization is outlined in Figure 9A, and the progress of the VL chain humanization is outlined in Figure 9B.

[0211] Example 19. Production and biophysical evaluation of a partially humanized version of anti-CD40 clone 5B9 Different humanized versions of clone 5B9 scFv were produced as monospecific DNA constructs by attaching the scFv sequence to the N-terminus of the wild-type IgG1 Fc region. After transient expression and purification of sufficient quantities of the constructs, these constructs were characterized for expression, thermal stability by differential scanning fluorimetry (DSF), and either binding affinity to human CD40 ECD by SPR (as described in Example 20) or cell binding (shown in Example 21).

[0212] The midpoint of the first melting transition (T m1 ) was measured using DSF. m1 was used to reflect the temperature required to unfold the first, or least stable, binding domain in the construct. DSF was performed using an Uncle instrument from Unchained Laboratories. Samples were analyzed at 1 mg / mL in PBS using intrinsic fluorescence (no additional dye was used to assess protein unfolding). As shown in Table 16, all constructs exhibited T > 65°C. m1 and exhibits high thermal stability.

[0213] Furthermore, expression levels varied throughout the humanization process, ranging from 54.3 to 424.5 μg / mL, with the highest expression exceeding 200 μg / mL. The significantly higher expression levels combined with the Tm values ​​suggest that these variants possess stability and solubility properties that may be beneficial for therapeutic protein drugs. Table 16. Summary of expression, thermostability, and affinity of partially humanized anti-CD40 variants of clone 5B9 [Table 15]

[0214] Example 20. Methodology for determining binding affinity of anti-CD40 binding domains using surface plasmon resonance SPR binding affinity testing of anti-CD40 binding domains to recombinant monomeric human CD40 ectodomain (ECD) was performed on a Biacore T200 system in HBS-EP+ containing 0.1% BSA buffer at 25°C. Mouse anti-human IgG (Cytiva, BR100839) at 25 μg / ml in 10 mM sodium acetate, pH 5.0, was immobilized to each flow cell of a CM5 research-grade sensor chip (Cytiva, BR100530) at a density of 4,000 response units (RU) using standard amine coupling chemistry. Approximately 40 nM of each anti-CD40 monospecific or bispecific protein in HBS-EP+ containing 0.1% BSA buffer was captured onto the flow cell with the immobilized anti-human IgG for 20 seconds at a flow rate of 10 μl / min, leaving one unmodified flow cell surface as a reference. Using single-cycle kinetics mode, CD40 ECD at different concentrations, ranging from 8 nM to 648 nM, was injected sequentially through each flow cell at 30 μl / min for up to 400 s, followed by a 600 s dissociation period. Regeneration was achieved by injecting 3 M MgCl2 at a flow rate of 30 μl / min for 30 s, followed by a 1-min stabilization period in HBS-EP+ containing 0.1% BSA buffer.

[0215] The sensorgrams obtained from kinetic SPR measurements were analyzed by double subtraction. The signal from the reference flow cell was subtracted from the analyte binding response obtained from the flow cell with immobilized or captured ligand. The buffer reference response was then averaged from multiple injections. The averaged buffer reference response was then subtracted from the analyte binding response, and the final double-referenced data was analyzed using Biacore T200 evaluation software (v2.0, Cytiva), and the data were globally fitted to derive kinetic parameters. All sensorgrams were fitted using a simple one-to-one binding model.

[0216] Several monospecific and bispecific anti-CD40 proteins were evaluated for binding affinity to monomeric human CD40 ECD using SPR (see Tables 15, 16, and 19).

[0217] Example 21. Evaluation of anti-CD40 binding to CD40-expressing CHO cell lines during humanization Throughout the humanization of the anti-CD40 clones 5B9 and 1D5 binding domains, these constructs were evaluated for retained on-cell binding similar to the parent domain. Each successive set of changes was tested by flow cytometry using human CHOK1SV / CD40 cells as described above. Humanization of clone 5B9 began first and is shown as part of Figures 10A and 10B. All 5B9 modifications retained CD40 binding similar to the original. Figures 10B-10E all evaluate 1D5 humanization. Reduced 1D5 binding to CD40 is shown for the modification in Figure 10C, but was restored for the modifications shown in Figures 10D and 10E.

[0218] Example 22. Binding of CD40-binding molecules to human CD40- and PD-L1-expressing tumor cell lines Binding studies were used to confirm binding of the anti-PD-L1 x anti-CD40 bispecific variants to human tumor cell lines known to express CD40 and / or PD-L1. Flow cytometry was used to examine the binding of each bispecific end. As shown in Figure 11A, binding to Daudi tumor cells varied significantly depending on the bispecific. In contrast, there was minimal difference in binding between the variants on MDA-MB-231 (Figure 11B).

[0219] Example 23. Assembly and characterization of bispecific anti-PD-L1 x anti-CD40 constructs containing mutations that improve manufacturability to the anti-CD40 scFv A subset of PD-L1 and CD40 binding domains were combined into bispecific proteins. Individual binding domains were amplified by PCR and assembled with an Fc-encoding DNA fragment and a linearized expression vector using standard molecular biology techniques. After analysis of experimental data and in silico modeling of the CD40 binding domain in the parent bispecific PC401003, several variants were constructed and tested. Mutations were designed to improve manufacturability characteristics and identified using BioLuminate (Schroedinger Release 2022-4: BioLuminate, Schroedinger, LLC, New York, NY, 2021). The parent PC401003 amino acid sequence was mutated with single or double point mutations listed in Table 17. The bispecific molecules PC401119, PC401120, PC401122, PC401124, PC401128, PC401129, PC401132 and PC401133 (see Table 1) represent a set of CD40 domain variants with the most advantageous mutations.

[0220] Following transient transfection and purification using the methods described above, these additional bispecific proteins were examined for the effect of incorporating additional mutations into the anti-CD40 scFv. The orientation was kept constant (anti-PD-L1 at the N-terminus, anti-CD40 at the C-terminus) and both the PD-L1 scFv and Fc sequences remained unchanged. Table 17. Description of bispecific constructs evaluated for manufacturable mutations in anti-CD40 scFv [Table 16]

[0221] Comparison of the constructs shows that transient expression levels are affected by all stabilizing mutations compared to the parent PC401003. Within this set, PC401120 and PC401122 retained the highest expression levels. Preparative SEC was performed following a Protein A affinity capture step to remove high molecular weight aggregates (HMW) while simultaneously buffer-exchanging the samples into PBS. Samples of each construct were analyzed by analytical HPLC to assess product homogeneity. All constructs in Table 18 had high purity levels (>93% MP) with negligible HMW products detected by HPLC. Table 18. Expression levels and purity of bispecific anti-PD-L1 x anti-CD40 constructs containing stabilizing mutations [Table 17]

[0222] For these constructs, T m1 (midpoint of first melting transition) and T agg (onset temperature of aggregation based on dynamic light scattering) was measured using an Uncle instrument from Unchained Labs. Most of the constructs shown in Table 24, including the parent PC401003, had a T above 60°C. m1 and T agg Only PC401129 had a T value of 56.2°C, indicating high thermal stability. m1 PC401120 had almost completely higher T than its parent PC401003. m1 had the following characteristics:

[0223] Using the BIACORE T200 SPR system, the affinity of a subset of these constructs for human CD40 ECD was determined using the methods described above. Monovalent binding affinity was determined by capturing the bispecific constructs on a chip and injecting multiple concentrations of the target monovalent ECD. The affinity of the CD40 scFvs was not measurably affected by the stabilizing mutations, as all KDs remained in the low nM range (Table 19). Further analysis of binding affinity to CD40 and PD-L1 by on-cell binding is shown in Example 24. Table 19. T of bispecific anti-PD-L1 x anti-CD40 constructs containing stabilizing mutations m1 and T agg value and binding affinity to human CD40 ECD [Table 18] JPEG2026501724000031.jpg27159

[0224] Example 24. Binding of anti-PD-L1 x anti-CD40 bispecific proteins to various cell lines The stabilized CD40-binding domains were evaluated for retained binding to CD40-expressing cell lines using flow cytometry. As shown in Figures 12A-12E, the majority of sequence modifications had little effect on the original binding of the humanized 1D5 construct. Figures 12A and 12B are graphical representations of binding to CHOK1SV expressing either human or cynomolgus monkey CD40, respectively. The fully optimized CD40-binding domains were evaluated for binding to Daudi cells, a tumor cell line that endogenously expresses CD40. Figure 12C shows that PC401120 binds slightly less than either PC401119 or PC401122. To ensure that binding to PD-L1 was not negatively altered, binding to CHOK1SV / PD-L1 was retested and found to be unchanged (Figure 12D). Furthermore, the complete construct was assessed for non-specific binding to the parental CHOK1SV (Fig. 12E), but remained at background levels comparable to the parental 1D5 construct PC401003.

[0225] Example 25. Functional reporter assay evaluating anti-PD-L1 x anti-CD40 bispecific proteins To compare the activity of anti-PD-L1 x anti-CD40 bispecific proteins, two luciferase reporter lines were used in separate assays. PD-L1-expressing cells were used to bind and induce crosslinking of the other anti-receptor binding domain of the bispecific. To quantify PD-L1 / PD-1 blocking activity, the human PD-1 NFAT luciferase reporter Jurkat line was incubated with CHO target cells expressing the TCR activator PD-L1. As shown in Figure 13A, the PD-1 / PD-L1 blocking activity of all bispecifics tested was very similar and unaffected by sequence modifications of the anti-CD40 binding domain. Blocking activity was similar to that of the atezolizumab analog control (PDL01029). The CD40 control sample alone did not produce an enhanced luminescent signal.

[0226] Conversely, to examine CD40 activity, human CD40 / NFκB HEK293 luciferase reporter cells were incubated with PD-L1-expressing target cells. As shown in Figure 13B, the CD40 activity of all bispecifics tested was indistinguishable, again unaffected by sequence modifications in the anti-CD40 binding domain. CD40 reporter assays demonstrate that the functional activity of our bispecifics is between that of analogs of celicrelumab (CD401085) and MEDI7526 (PDL01127). The negative control antibody alone failed to induce CD40 activity. Figure 13C demonstrates that the bispecifics require PD-L1 cross-linking for CD40 signaling, as CD40-mediated luminescence is not increased when parental CHOK1SV cells are used. In contrast, analogs of celicrelumab and MEDI7526, which do not require cross-linking to function, induce similar levels of CD40 signaling in the absence of PD-L1-expressing cells.

[0227] Example 26. Functional reporter assay evaluating anti-PD-L1 x anti-CD40 bispecific proteins To compare the activity of the optimized anti-PD-L1 x anti-CD40 bispecific proteins, live-cell imaging was used to demonstrate tumor cell killing. First, the squamous cell carcinoma line SCC152 (ATCC) was transduced to express several proteins. SCC152 cells were transduced with the lentivirus Nuclight Orange (NLO, Sartorius) and selected using puromycin. Expanded cells were then transduced with in-house generated lentiviral human PD-L1 and selected with zeocin and puromycin. Finally, SCC152 were transduced with lentiviral EBV proteins (Vectorbuilder) and selected using neocin, zeocin, and puromycin. Triple-positive cells were shown to be >99% positive by flow cytometry (Figure 14A).

[0228] To set up the tumor cell killing assay, several primary cell subsets were generated. M2 macrophages, immature DCs, and EBV-specific T cells were all prepared separately prior to setting up the assay. EBV-expressing SCC152 tumor cells were treated with 10 ng / ml IFNg two weeks prior to the start of the assay. The following day, SCC152 tumor cells were treated with 130 KeV for 10 minutes using a Faxitron x-ray irradiator. Ten million PBMCs (Bloodworks) were plated with 1e6 irradiated SCC152 cells. Cytokines were added to promote T cell proliferation (IL2 (10 ng / ml), IL7 (10 ng / ml), IL15 (10 ng / ml), and IL21 (10 ng / ml)). T cells were expanded in fresh medium and cytokines for two weeks, then harvested and assessed for tetramer staining and IFNg production.

[0229] One week before assay setup, M2 monocytes and immature DCs were generated from adherent PBMCs (Bloodworks). For M2 monocytes, 100 ng / ml M-CSF (Peprotech) was added on day 0. IL4 (50 ng / ml) and IL10 (50 ng / ml) were then added to the feed medium on day 5 to polarize the cells. For DCs, IL4 (25 ng / ml) and GM-CSF (100 ng / ml) were added to the cultures on days 0 and 5. All cells were harvested on day 7 for use in the killing assay.

[0230] The day before assay setup, 10,000 adherent SCC152 cells (expressing EBV, NLO, and PD-L1) were seeded into a 96-well plate and incubated overnight. The medium was then removed and replaced with two titrations of PD-L1 x CD40 test or control molecules. Immature DCs, macrophages, and T cells were mixed and transferred to the assay plate at final cell numbers of 500, 500, and 1,000 per well, respectively. The plate was then transferred to an Incucyte reader (Sartorius) and imaged every 8 hours over the course of 6 days. Analysis was completed after the 6-day period.

[0231] Figure 14B graphically depicts the reduction in the number of live SCC152 tumor cells with 0.25 nM of test or control molecules. The CD40 control molecule, PC401242, was unable to enhance tumor cell killing. Both PD-L1 (PC401015) and the parent bispecific (PC401003) were able to enhance SCC152 killing, but were less effective than the optimized molecules. PC401122 was slightly better than the parent molecule, but both PC401119 and PC401120 were superior in enhancing tumor killing.

[0232] Example 27. Epitope mapping of anti-CD40 binding domain 1D5 The domain mapping principles described in Example 11 were extended and further refined to map the epitope of the anti-CD40 antibody 1D5. Both the fully murine and the final humanized versions of the 1D5 binding domain in scFv format were examined, with identical results.

[0233] For a protein alignment of all sequences used for epitope mapping, see Figure 15 (Protein sequence alignment of CD40 extracellular domains used for epitope mapping of the 1D5 antibody). In Example 11, we showed that 1D5 binds to the region between His122 and Arg193. Using the published structure of human CD40, pdb 7P3I_1, we designed three sets of spatially colocalized amino acids in domains 3 and 4, labeled 31, 32, and 33. The amino acids in these sets, representing the human CD40 sequences, were mutated to their mouse CD40 counterparts. When expressed on the surface of CHO cells and tested by flow cytometry using the 1D5 binding domain, only one construct, hu / mu33_CD40_ECD, lost binding (see Figure 15). Next, we individually mutated all nine human amino acids from construct 33 to their mouse sequences. Only one mutation, E144H, adversely affected binding. Based on the published structure, alanine scanning mutagenesis was performed on 13 selected residues spatially surrounding Glu144 (see Figure 15). The data are summarized in Table 20. The data confirmed a very strong contribution of the E144 residue, a strong contribution of F150, and significant contributions of residues F129, Q133, V138, S139, I142, P147, V148, F150, and K160 to binding of both the murine parental 1D5 binding domain and the humanized 1D5 binding domain. Table 20. Alanine scanning to identify key human CD40 residues involved in binding to the 1D5 antibody. [Table 19] Binding was normalized to the FLAG signal and values ​​shown are relative to huCD40.

[0234] Example 28. Surface plasmon resonance (SPR) experiments to confirm the abrogation of binding to Fcγ receptors by modified Fc domains The immunoglobulin constant domain (Fc) contained in certain bispecific anti-PD-L1 x anti-CD40 lead constructs incorporates modifications designed to prevent or reduce Fcg receptor binding compared to the wild-type immunoglobulin constant region (FcγR1, FcγRIIa, FcγRIIb, FcγRIIa, and FcγRIIIb), as described in patent WO2022178114A1. The modified immunoglobulin constant region comprises a human IgG1 CH2 domain containing the substitutions E233P, L234A, L235A, G237A, and K322A according to the EU numbering system, and a deletion of G236.

[0235] Binding of Fcg receptors to wild-type and modified immunoglobulin constant domain (Fc) protein fragments was measured by surface plasmon resonance at room temperature on a Biacore 8K instrument. The modified Fc protein fragments were directly immobilized onto the surface of a CM5 sensor chip using standard amine coupling chemistry to a surface density of approximately 1000 RU. A wild-type Fc fragment was similarly immobilized on the surface as a positive control and used as a comparison to assess binding reductions resulting from changes engineered into the amino acid sequence of the Fc region. Fcg receptors (purchased from R&D Systems) were diluted to either 100 nM (FcgRI) or 2 μM (all other Fcg receptors) in HBS-EP+ buffer and then injected over the prepared sensor chip surface at 30 μL / min for 60 seconds. The maximum RU value during the association phase of each injection was normalized to the wild-type Fcg result, and the calculated relative binding values ​​are reported in Table 21. The listed Fc modifications significantly reduced binding to all receptors tested, with some significant binding remaining to the Fcg RIIA and RIIB / C receptors. Table 21. Binding of wild-type and modified Fc fragments to different Fc receptors [Table 20]

[0236] Example 29. Surface plasmon resonance (SPR) experiments to confirm retention of binding affinity to fetal Fc receptor (FcRn) The immunoglobulin constant domain contained in certain bispecific anti-PD-L1 x anti-CD40 lead constructs incorporates modifications designed to prevent or reduce Fcg receptor binding compared to the wild-type immunoglobulin constant region, while maintaining expected fetal Fc receptor (FcRn) binding, as described in patent WO2022178114A1. The modified immunoglobulin constant region comprises a human IgG1 CH2 domain containing the substitutions E233P, L234A, L235A, G237A, and K322A according to the EU numbering system, and a deletion of G236.

[0237] SPR binding studies of bispecific anti-PD-L1 x anti-CD40 proteins containing modified Fc to human fetal Fc receptor (FcRn, complexed with beta-2-macroglobulin) were performed on a Biacore T200 system at 25°C in a running buffer of phosphate-buffered saline (PBS) containing 0.1% BSA and 0.05% polysorbate-20 adjusted to pH 6.0. Human FcRn / b2m at 10 μg / ml in 10 mM sodium acetate (pH 4.5) was immobilized to a CM5 sensor chip (Cytiva, BR100530) by direct amine coupling chemistry to a level of approximately 340 RU. A reference flow cell was left blank. Different concentrations of bispecific anti-PD-L1 x anti-CD40 proteins, ranging from 2 nM to 162 nM, diluted in running buffer were injected continuously for 180 seconds at 30 μl / min, followed by a 180-second dissociation period. Optimal regeneration was achieved by two 30-second injections of PBS containing 0.1% BSA and 0.05% polysorbate-20 adjusted to pH 7.4 at a flow rate of 30 μl / min, followed by 1 minute of running buffer stabilization.

[0238] Sensorgrams obtained from kinetic SPR measurements were analyzed by the double subtraction method. The signal from the reference flow cell was subtracted from the analyte binding response obtained from the flow cell with immobilized ligand. The buffer reference was subtracted from the analyte binding response, and the final double-referenced data was analyzed using Biacore T200 evaluation software (v2.0, Cytiva), and the data were globally fitted to derive kinetic parameters. All sensorgrams were fitted using the two-state reaction model described in Weirong Wang et al., Drug Meta Dispos. 2011 Sep;39(9):1469-77. All bispecific anti-PD-L1 x anti-CD40 proteins tested maintained the expected binding to FcRn.

[0239] Several bispecific anti-PD-L1 x anti-CD40 proteins were evaluated for binding affinity to the fetal Fc receptor (FcRn) using SPR (see Table 22). Table 22. SPR affinity measurements for fetal Fc receptor (FcRn) [Table 21]

[0240] Example 30. Evaluation of anti-CD40 bispecifics targeting either ROR1 or EGFR tumor-associated antigens This example demonstrates that the provided CD40 binding domain can be paired with various tumor-associated antigens and utilized to target any number of tumor types. Our humanized 1D5 CD40 binding domain was linked to either ROR1 or EGFR-specific binding domains. These tumor antigens are expressed on Kasumi-...

Claims

1. A PD-L1-binding polypeptide that specifically binds to human PD-L1, the polypeptide having, in order from amino terminus to carboxyl terminus or carboxyl terminus to amino terminus, (a) a first binding domain; (b) Hinge region (c) an immunoglobulin constant region, and (d) a second binding domain; the first binding domain is a PD-L1 binding domain and the second binding domain binds to an immunostimulatory protein; or The PD-L1-binding polypeptide, wherein the first binding domain binds to an immunostimulatory protein and the second binding domain is a PD-L1-binding domain.

2. the PD-L1 binding domain is (i) an immunoglobulin heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3; and (ii) the PD-L1-binding polypeptide of claim 1, comprising an immunoglobulin light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3.

3. 3. The PD-L1-binding polypeptide of claim 2, wherein the HCDR1 comprises SEQ ID NO: 1, the HCDR2 comprises SEQ ID NO: 2, the HCDR3 comprises SEQ ID NO: 3, the LCDR1 comprises SEQ ID NO: 5, the LCDR2 comprises SEQ ID NO: 6, and the LCDR3 comprises SEQ ID NO:

7.

4. 4. The PD-L1-binding polypeptide of claim 2 or 3, wherein the VH comprises SEQ ID NO:4 and the VL comprises SEQ ID NO:

8.

5. The PD-L1-binding polypeptide of any one of claims 1 to 4, wherein the PD-L1-binding domain comprises SEQ ID NO:

9.

6. The PD-L1-binding polypeptide of any one of claims 1 to 4, wherein the immunostimulatory protein is CD40, 4-1BB, CD3, or OX40.

7. A CD40-binding polypeptide that specifically binds to human CD40, the polypeptide having, in order from amino terminus to carboxyl terminus or carboxyl terminus to amino terminus, (a) a first binding domain; (b) Hinge region (c) an immunoglobulin constant region, and (d) a second binding domain; the first binding domain is a CD40 binding domain and the second binding domain binds to a tumor-associated antigen; or The CD40-binding polypeptide, wherein the first binding domain binds to a tumor-associated antigen and the second binding domain is a CD40-binding domain.

8. the CD40 binding domain is (i) an immunoglobulin heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3; and (ii) a CD40-binding polypeptide according to claim 7, comprising an immunoglobulin light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3.

9. (a) the HCDR1 comprises SEQ ID NO: 10, the HCDR2 comprises SEQ ID NO: 11, the HCDR3 comprises SEQ ID NO: 12, the LCDR1 comprises SEQ ID NO: 14, the LCDR2 comprises SEQ ID NO: 15, and the LCDR3 comprises SEQ ID NO: 16; or (b) the HCDR1 comprises SEQ ID NO: 10, the HCDR2 comprises SEQ ID NO: 23, the HCDR3 comprises SEQ ID NO: 12, the LCDR1 comprises SEQ ID NO: 14, the LCDR2 comprises SEQ ID NO: 15, and the LCDR3 comprises SEQ ID NO: 16; or (c) the HCDR1 comprises SEQ ID NO: 10, the HCDR2 comprises SEQ ID NO: 11, the HCDR3 comprises SEQ ID NO: 31, the LCDR1 comprises SEQ ID NO: 14, the LCDR2 comprises SEQ ID NO: 15, and the LCDR3 comprises SEQ ID NO: 16; or (d) the HCDR1 comprises SEQ ID NO: 10, the HCDR2 comprises SEQ ID NO: 23, the HCDR3 comprises SEQ ID NO: 31, the LCDR1 comprises SEQ ID NO: 14, the LCDR2 comprises SEQ ID NO: 15, and the LCDR3 comprises SEQ ID NO: 16; or (e) the HCDR1 comprises SEQ ID NO: 10, the HCDR2 comprises SEQ ID NO: 11, the HCDR3 comprises SEQ ID NO: 12, the LCDR1 comprises SEQ ID NO: 37, the LCDR2 comprises SEQ ID NO: 15, and the LCDR3 comprises SEQ ID NO: 16; or (f) the HCDR1 comprises SEQ ID NO: 10, the HCDR2 comprises SEQ ID NO: 11, the HCDR3 comprises SEQ ID NO: 12, the LCDR1 comprises SEQ ID NO: 41, the LCDR2 comprises SEQ ID NO: 15, and the LCDR3 comprises SEQ ID NO: 16; or (g) the HCDR1 comprises SEQ ID NO: 10, the HCDR2 comprises SEQ ID NO: 45, the HCDR3 comprises SEQ ID NO: 12, the LCDR1 comprises SEQ ID NO: 14, the LCDR2 comprises SEQ ID NO: 15, and the LCDR3 comprises SEQ ID NO: 16; or (h) the HCDR1 comprises SEQ ID NO: 10, the HCDR2 comprises SEQ ID NO: 49, the HCDR3 comprises SEQ ID NO: 12, the LCDR1 comprises SEQ ID NO: 14, the LCDR2 comprises SEQ ID NO: 15, and the LCDR3 comprises SEQ ID NO: 16; or (i) The CD40-binding polypeptide of claim 8, wherein the HCDR1 comprises SEQ ID NO: 10, the HCDR2 comprises SEQ ID NO: 23, the HCDR3 comprises SEQ ID NO: 31, the LCDR1 comprises SEQ ID NO: 37, the LCDR2 comprises SEQ ID NO: 15, and the LCDR3 comprises SEQ ID NO:

16.

10. (a) the VH comprises SEQ ID NO: 13 and the VL comprises SEQ ID NO: 17; or (b) the VH comprises SEQ ID NO: 24 and the VL comprises SEQ ID NO: 17; or (c) the VH comprises SEQ ID NO: 27 and the VL comprises SEQ ID NO: 28; or (d) the VH comprises SEQ ID NO: 32 and the VL comprises SEQ ID NO: 17; or (e) the VH comprises SEQ ID NO: 34 and the VL comprises SEQ ID NO: 17; or (f) the VH comprises SEQ ID NO: 13 and the VL comprises SEQ ID NO: 38; or (g) the VH comprises SEQ ID NO: 13 and the VL comprises SEQ ID NO: 42; or (h) the VH comprises SEQ ID NO: 46 and the VL comprises SEQ ID NO: 17; or (i) the VH comprises SEQ ID NO: 50 and the VL comprises SEQ ID NO: 17; or (j) the VH comprises SEQ ID NO: 484 and the VL comprises SEQ ID NO: 485; or (k) The CD40-binding polypeptide of claim 8 or 9, wherein the VH comprises SEQ ID NO: 484 and the VL comprises SEQ ID NO:

28.

11. 11. The CD40-binding polypeptide of any one of claims 7 to 10, wherein the CD40-binding domain comprises SEQ ID NO: 18, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 486, or SEQ ID NO:

488.

12. The CD40-binding polypeptide according to any one of claims 7 to 11, wherein the tumor-associated antigen is PD-L1, ROR1, or EGFR.

13. A binding polypeptide that specifically binds to human PD-L1 and human CD40, the binding polypeptide having, in order from amino terminus to carboxyl terminus or carboxyl terminus to amino terminus, (a) a first binding domain; (b) Hinge region (c) an immunoglobulin constant region, and (d) a second binding domain; the first binding domain is a PD-L1 binding domain and the second binding domain is a CD40 binding domain; or The binding polypeptide, wherein the first binding domain is a CD40-binding domain and the second binding domain is a PD-L1-binding domain.

14. the PD-L1 binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; 14. The binding polypeptide of claim 13, wherein the CD40 binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 11, and an HCDR3 comprising SEQ ID NO: 12, and a VL comprising an LCDR1 comprising SEQ ID NO: 14, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO:

16.

15. the PD-L1 binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; 14. The binding polypeptide of claim 13, wherein the CD40 binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 23, and an HCDR3 comprising SEQ ID NO: 12, and a VL comprising an LCDR1 comprising SEQ ID NO: 14, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO:

16.

16. the PD-L1 binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; 14. The binding polypeptide of claim 13, wherein the CD40 binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 11, and an HCDR3 comprising SEQ ID NO: 31, and a VL comprising an LCDR1 comprising SEQ ID NO: 14, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO:

16.

17. the PD-L1 binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; 14. The binding polypeptide of claim 13, wherein the CD40 binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 23, and an HCDR3 comprising SEQ ID NO: 31, and a VL comprising an LCDR1 comprising SEQ ID NO: 14, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO:

16.

18. the PD-L1 binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; 14. The binding polypeptide of claim 13, wherein the CD40 binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 11, and an HCDR3 comprising SEQ ID NO: 12, and a VL comprising an LCDR1 comprising SEQ ID NO: 37, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO:

16.

19. the PD-L1 binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; 14. The binding polypeptide of claim 13, wherein the CD40 binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 11, and an HCDR3 comprising SEQ ID NO: 12, and a VL comprising an LCDR1 comprising SEQ ID NO: 41, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO:

16.

20. the PD-L1 binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; 14. The binding polypeptide of claim 13, wherein the CD40 binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 45, and an HCDR3 comprising SEQ ID NO: 12, and a VL comprising an LCDR1 comprising SEQ ID NO: 14, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO:

16.

21. the PD-L1 binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; 14. The binding polypeptide of claim 13, wherein the CD40 binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 49, and an HCDR3 comprising SEQ ID NO: 12, and a VL comprising an LCDR1 comprising SEQ ID NO: 14, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO:

16.

22. the PD-L1 binding domain comprises an immunoglobulin heavy chain variable region (VH) comprising an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3, and an immunoglobulin light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 5, an LCDR2 comprising SEQ ID NO: 6, and an LCDR3 comprising SEQ ID NO: 7; 14. The binding polypeptide of claim 13, wherein the CD40 binding domain comprises a VH comprising an HCDR1 comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 23, and an HCDR3 comprising SEQ ID NO: 31, and a VL comprising an LCDR1 comprising SEQ ID NO: 37, an LCDR2 comprising SEQ ID NO: 15, and an LCDR3 comprising SEQ ID NO:

16.

23. (a) the PD-L1-binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40-binding domain comprises a VH comprising SEQ ID NO: 13 and a VL comprising SEQ ID NO: 17; or (b) the PD-L1-binding domain comprises a VH comprising SEQ ID NO:4 and a VL comprising SEQ ID NO:8, and the CD40-binding domain comprises a VH comprising SEQ ID NO:24 and a VL comprising SEQ ID NO:17; or (c) the PD-L1-binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40-binding domain comprises a VH comprising SEQ ID NO: 27 and a VL comprising SEQ ID NO: 28; or (d) the PD-L1-binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40-binding domain comprises a VH comprising SEQ ID NO: 32 and a VL comprising SEQ ID NO: 17; or (e) the PD-L1-binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40-binding domain comprises a VH comprising SEQ ID NO: 34 and a VL comprising SEQ ID NO: 17; or (f) the PD-L1-binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40-binding domain comprises a VH comprising SEQ ID NO: 13 and a VL comprising SEQ ID NO: 38; or (g) the PD-L1-binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40-binding domain comprises a VH comprising SEQ ID NO: 13 and a VL comprising SEQ ID NO: 42; or (h) the PD-L1-binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40-binding domain comprises a VH comprising SEQ ID NO: 46 and a VL comprising SEQ ID NO: 17; or (i) the PD-L1-binding domain comprises a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 8, and the CD40-binding domain comprises a VH comprising SEQ ID NO: 50 and a VL comprising SEQ ID NO: 17; or (j) the PD-L1 binding domain comprises a VH comprising SEQ ID NO:4 and a VL comprising SEQ ID NO:8, and the CD40 binding domain comprises a VH comprising SEQ ID NO:484 and a VL comprising SEQ ID NO:485; or (k) The binding polypeptide of any one of claims 13-22, wherein the PD-L1 binding domain comprises a VH comprising SEQ ID NO:4 and a VL comprising SEQ ID NO:8, and the CD40 binding domain comprises a VH comprising SEQ ID NO:484 and a VL comprising SEQ ID NO:

28.

24. (a) the PD-L1-binding domain comprises SEQ ID NO: 9 and the CD40-binding domain comprises SEQ ID NO: 18; or (b) the PD-L1 binding domain comprises SEQ ID NO: 9 and the CD40 binding domain comprises SEQ ID NO: 25; or (c) the PD-L1 binding domain comprises SEQ ID NO: 9 and the CD40 binding domain comprises SEQ ID NO: 29; or (d) the PD-L1 binding domain comprises SEQ ID NO: 9 and the CD40 binding domain comprises SEQ ID NO: 33; or (e) the PD-L1 binding domain comprises SEQ ID NO: 9 and the CD40 binding domain comprises SEQ ID NO: 35; or (f) the PD-L1 binding domain comprises SEQ ID NO: 9 and the CD40 binding domain comprises SEQ ID NO: 39; or (g) the PD-L1 binding domain comprises SEQ ID NO: 9 and the CD40 binding domain comprises SEQ ID NO: 43; or (h) the PD-L1 binding domain comprises SEQ ID NO: 9 and the CD40 binding domain comprises SEQ ID NO: 47; or (i) the PD-L1 binding domain comprises SEQ ID NO: 9 and the CD40 binding domain comprises SEQ ID NO: 51; or (j) the PD-L1 binding domain comprises SEQ ID NO: 9 and the CD40 binding domain comprises SEQ ID NO: 486; or (k) The binding polypeptide of any one of claims 13-23, wherein the PD-L1 binding domain comprises SEQ ID NO: 9 and the CD40 binding domain comprises SEQ ID NO:

488.

25. 25. The binding polypeptide of any one of claims 13-24, wherein the binding polypeptide comprises SEQ ID NO:19, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:280, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:487, or SEQ ID NO:

489.

26. 26. The binding polypeptide of any one of claims 1 to 25, wherein the hinge is an immunoglobulin hinge.

27. 27. The binding polypeptide of any one of claims 1 to 26, wherein the immunoglobulin constant region comprises the immunoglobulin CH2 and CH3 domains of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD.

28. 28. The binding polypeptide of claim 27, wherein the immunoglobulin constant region comprises a human IgGl CH2 domain comprising amino acid substitutions at one or more of the following residues according to the EU numbering system: E233, L234, L235, G236, G237, E318, K320, K322.

29. 29. The binding polypeptide of claim 28, wherein the amino acid substitution at residue E233 is E233P.

30. 29. The binding polypeptide of claim 28, wherein the amino acid substitution at residue L234 is selected from the group consisting of L234A and L234V.

31. 29. The binding polypeptide of claim 28, wherein the amino acid substitution at residue L235 is L235A.

32. 29. The binding polypeptide of claim 28, wherein the amino acid substitution at residue G237 is G237A.

33. 29. The binding polypeptide of claim 28, wherein the amino acid substitution at E318 is E318A.

34. 29. The binding polypeptide of claim 28, wherein the amino acid substitution at K320 is K320A.

35. 29. The binding polypeptide of claim 28, wherein the amino acid substitution at K322 is K322A.

36. 28. The binding polypeptide of claim 27, wherein residue G236 (according to the EU numbering system) is deleted.

37. A dimeric protein comprising a binding polypeptide according to any one of claims 1 to 36.

38. 37. The dimeric protein of claim 36, wherein the dimeric protein is a homodimer.

39. 37. A composition comprising a binding polypeptide of any one of claims 1 to 36 and a pharmaceutically acceptable carrier, diluent or excipient.

40. 39. A composition comprising the dimeric protein of claim 37 or 38 and a pharmaceutically acceptable carrier, diluent or excipient.

41. A nucleic acid molecule comprising a nucleotide sequence encoding a binding polypeptide of any one of claims 1 to 36.

42. 42. The nucleic acid molecule of claim 41, wherein the nucleotide sequence comprises SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:259, or SEQ ID NO:

260.

43. 43. An expression vector comprising the nucleic acid molecule of claim 41 or 42.

44. 44. A recombinant host cell comprising the nucleic acid molecule of claim 41 or 42 or the expression vector of claim 43.

45. 1. A method for producing a binding polypeptide, comprising: Culturing the recombinant host cell of claim 44 under conditions in which said nucleic acid molecule is expressed, thereby producing said binding polypeptide; and isolating the binding polypeptide from the host cell or culture.

46. 41. A method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a binding polypeptide of any one of claims 1 to 36, a dimeric protein of claim 37 or 38, or a composition of claim 39 or 40.

47. 41. A method for treating a symptom of cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a binding polypeptide of any one of claims 1 to 36, a dimeric protein of claim 37 or 38, or a composition of claim 39 or 40.

48. 48. The method of claim 46 or 47, wherein the cancer is a solid tumor.

49. The method of any one of claims 46 to 48, wherein the cancer expresses PD-L1.

50. 49. The method of any one of claims 46 to 48, wherein the cancer is head and neck cancer, melanoma, lung cancer, brain cancer, thymus cancer, breast cancer, liver cancer, pancreatic cancer, kidney cancer, ovarian cancer, bladder cancer, gastrointestinal cancer, or colon cancer.

51. A binding polypeptide according to any one of claims 1 to 36, a dimeric protein according to claim 37 or 38, or a composition according to claim 39 or 40, for use as a medicament.

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