EA043064B1 - ANTIBODIES SPECIFICALLY BINDING TO PD-1 AND METHODS OF THEIR APPLICATION - Google Patents

Description

The currently pending application contains a sequence listing provided in electronic form in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy of this listing created on May 30, 2018 is called

JBI5131WOPCT_ST25.txt and is 220 kB in size.

Scope of the invention

The invention relates to antibodies that specifically bind to PD-1, polynucleotides encoding antibodies or antigen-binding fragments, as well as methods for obtaining and using the above.

Prerequisites for the creation of the invention

PD-1 (programmed cell death protein-1; PDCD1) is an inhibitory receptor that belongs to the CD28/CTLA-4 family. PD-1 is a type I transmembrane glycoprotein that contains one extracellular domain and one cytoplasmic domain containing an immunoreceptor inhibitory tyrosine motif (ITIM) and an immunoreceptor switching tyrosine motif (ITSM). PD-1 is expressed on activated T cells, B cells, NK cells and thymocytes, and on resting memory T cells, including follicular helper T cells (T _FH ) and peripheral helper T cells (T _PH ). PD-1, after interacting with its PD-L1 or PD-L2 ligands, suppresses T cell function through multiple mechanisms (Pauken & Wherry (2015) Trends in Immunology 36(4): 265-276). Interaction with PD-1 directly inhibits T cell receptor (TCR) signaling through co-occurrence with TCR and subsequent induction of dephosphorylation of proximal TCR signaling molecules, inhibition of the Ras/MEK/ERK pathway leading to inhibition of cell cycle advancement and T cell proliferation, growth inhibition and cell survival and reprogramming of T cell metabolism by downregulating the PI3K/AKT pathway leading to an increase in the transcription factor BATF and modulation of regulatory T cell development, maintenance and function. It has also been suggested that PD-1 increases T cell motility and limits the duration of interaction between T cells and target cells, thus reducing the degree of T cell activation (Honda et al. (2014) Immunity 40 (2): 235 -47).

Studies in PD-1 deficient mice have shown that this pathway is important for both central and peripheral tolerance. PD-1 deficient mice derived from the C57Bl/6 line may develop spontaneous symptoms of autoimmune diseases, including autoantibody production, glomerulonephritis, and arthritis (Nishimura et al., Immunity 1999). These data indicate that PD-1 down-regulates immune responses.

Monoclonal antibodies to PD-1 and PD-L1 are approved for the treatment of cancers such as advanced melanoma, advanced non-small cell lung cancer, and classic Hodgkin's lymphoma. PD-1 has been found to be upregulated in many different types of tumors and is able to inhibit tumor-infiltrating PD-1+ T cells. Antagonistic monoclonal antibodies to PD-1 or PD-L1 reverse this suppression, allowing T cell activation and tumor attack. Thus, immune checkpoint blockade provides a way to enhance antitumor immune responses.

Despite the availability of biological anti-inflammatory therapeutics, there remains a need for improved anti-inflammatory drugs that can effectively suppress inflammation in the treatment of various immune disorders, such as rheumatoid arthritis, in which a significant proportion of patients still do not have an adequate response to therapy.

Statement of the Invention

The invention provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, containing the heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, the light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3 having the sequences SEQ ID NOs: 2, 165, 4, 166, 6 and 7, respectively.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 2, 3, 4, 5, 6 and 7, respectively; heavy chain variable region (VH) with SEQ ID NO: 8, 9 or 10 and VL with SEQ ID NO: 14, 15 or 16; and/or a heavy chain (HC) of SEQ ID NO: 20, 21 or 22 and a light chain (LC) of SEQ ID NO: 26, 27 or 28.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 2, 145, 4, 5, 6 and 7, respectively; VH with SEQ ID NO: 140 and VL with SEQ ID NO: 16; and/or HC with SEQ ID NO: 150 and LC with SEQ ID NO: 28.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 2, 146, 4, 5, 6 and 7, respectively; VH with SEQ ID NO: 141 and VL with SEQ ID NO: 16; and/or HC with SEQ ID NO: 151 and LC with SEQ ID NO: 28.

The invention also provides an isolated antibody that specifically binds to PD-1,

- 1 043064 or antigen-binding fragment containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and

LCDR3 with SEQ ID NO: 2, 147, 4, 5, 6 and 7, respectively; VH with SEQ ID NO: 142 and VL with SEQ ID NO: 16;

and/or HC with SEQ ID NO: 152 and LC with SEQ ID NO: 28.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 2, 3, 4, 148, 6, and 7, respectively; VH of SEQ ID NO: 10 and VL of SEQ ID NO: 143; and/or HC with SEQ ID NO: 22 and LC with SEQ ID NO: 153.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOS: 2, 3, 4, 149, 6 and 7, respectively; VH of SEQ ID NO: 10 and VL of SEQ ID NO: 144; and/or HC with SEQ ID NO: 22 and LC with SEQ ID NO: 154.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 2, 145, 4, 148, 6 and 7, respectively; VH of SEQ ID NO: 140 and VL of SEQ ID NO: 143; and/or HC with SEQ ID NO: 150 and LC with SEQ ID NO: 153.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 2, 146, 4, 148, 6 and 7, respectively; VH of SEQ ID NO: 141 and VL of SEQ ID NO: 143; and/or HC with SEQ ID NO: 151 AND LC with SEQ ID NO: 153.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 2, 147, 4, 148, 6 and 7, respectively; VH of SEQ ID NO: 142 and VL of SEQ ID NO: 143; and/or HC with SEQ ID NO: 152 AND LC with SEQ ID NO: 153.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOS: 2, 145, 4, 149, 6 and 7, respectively; VH with SEQ ID NO: 140 and VL with SEQ ID NO: 144; and/or HC with SEQ ID NO: 150 AND LC with SEQ ID NO: 154.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOS: 2, 146, 4, 149, 6 and 7, respectively; VH of SEQ ID NO: 141 and VL of SEQ ID NO: 144; and/or HC with SEQ ID NO: 151 AND LC with SEQ ID NO: 154.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 2, 147, 4, 149, 6 or 7, respectively, VH of SEQ ID NO: 142 and VL of SEQ ID NO: 144; and/or HC with SEQ ID NO: 152 AND LC with SEQ ID NO: 154.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3 having sequence SEQ ID NO: 32, 124, 40, 41, 42 and 43, respectively.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: SEQ ID NO: 32, 33, 40, 41, 42 and 43, respectively; VH with SEQ ID NO: 44 or 45 and VL with SEQ ID NO: 60, 61 or 62; and/or HC with SEQ ID NO: 66 or 67 and LC with SEQ ID NO: 82, 83 or 84.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOS: 32, 34, 40, 41, 42 and 43, respectively; VH with SEQ ID NO: 46 and VL with SEQ ID NO: 61; and/or HC with SEQ ID NO: 68 and LC with SEQ ID NO: 83.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 32, 35, 40, 41, 42 and 43, respectively; VH with SEQ ID NO: 47 and VL with SEQ ID NO: 61; and/or HC of SEQ ID NO: 69 and LC of SEQ ID NO: 83.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 32, 36, 40, 41, 42 and 43, respectively; VH of SEQ ID NO: 48 and VL of SEQ ID NO: 61; and/or HC with SEQ ID NO: 70 and LC with SEQ ID NO: 83.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOS: 32, 37, 40, 41, 42 and 43, respectively; VH of SEQ ID NO: 49 and VL of SEQ ID NO: 61; and/or HC with SEQ ID NO: 71 and LC with SEQ ID NO: 83.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and

- 2 043064

LCDR3 with SEQ ID NO: 32, 38, 40, 41, 42 and 43, respectively; VH with SEQ ID NO: 50 and VL with SEQ ID NO: 61;

and/or HC of SEQ ID NO: 72 and LC of SEQ ID NO: 83.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOS: 32, 39, 40, 41, 42 and 43, respectively; VH with SEQ ID NO: 51 and VL with SEQ ID NO: 61; and/or HC of SEQ ID NO: 73 and LC of SEQ ID NO: 83.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementarity determining region 1 (LCDR1), LCDR2 and lCdR3 having sequence SEQ ID NO: 88, 89, 90, 91, 92 and 93, respectively.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof competes for binding to PD-1 with an antibody of the invention.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to the same PD-1 epitope as the antibody of the invention.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, wherein the antibody VH and antibody VL or antibody HC and antibody LC are encoded by the specific polynucleotides described herein.

The invention also provides a polynucleotide encoding an antibody of the invention.

The invention also provides a vector containing at least one polynucleotide according to the invention.

The invention also provides a host cell containing the vector of the invention.

The invention also provides a method for producing an antibody or antigen-binding fragment of the invention, comprising culturing a host cell of the invention under conditions that express the antibody or antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment thereof.

The invention also provides a pharmaceutical composition containing an antibody or antigen-binding fragment of the invention.

The invention also provides a kit containing the antibody or antigen-binding fragment of the invention.

The invention also provides a method for suppressing the activation of a PD-1 expressing T cell in a patient, comprising administering to the patient an isolated antibody or an antigen-binding fragment thereof according to the invention for a time period sufficient to suppress the activation of a PD-1 expressing T cell.

The invention also provides a method for reducing an immune response, comprising administering to a patient in need thereof a therapeutically effective amount of an isolated antibody or antigen-binding fragment thereof according to the invention for a time period sufficient to reduce the immune response.

The invention also provides a method for treating an immune disorder, comprising administering to a patient in need thereof a therapeutically effective amount of an isolated antibody or antigen-binding fragment thereof according to the invention for a time period sufficient to treat the immune disorder.

The invention also provides an anti-idiotypic antibody that specifically binds to an antibody or antigen-binding fragment thereof of the invention.

The invention also provides an immunoconjugate containing an antibody or antigen-binding fragment of the invention conjugated to a heterologous molecule.

Brief description of graphic materials

In FIG. 1A shows that the selected generated antibodies inhibited T cell activation in a cytomegalovirus (CMV) secondary response assay with an inhibition level greater than 50% or higher. CNTO3930: isotype control. PD1B199: Antagonistic monoclonal antibody (mAb) to PD-1.

In FIG. 1B shows that the selected resulting antibodies inhibited T cell activation in the secondary CMV response assay with an inhibition level greater than 50% or greater. CNTO3930: isotype control. PD1B199: Antagonistic mAb to PD-1.

In FIG. 2A for mAb line PD1B505 shows the alignment of the VH regions of PD1H93 (SEQ ID NO: 8), PD1H384 (SEQ ID NO: 9), PD1H405 (SEQ ID NO: 10), PD1H585 (SEQ ID NO: 140), PD1H586 (SEQ ID NO: 140), NO: 141) and PD1H587 (SEQ ID NO: 142). Complementarity determining regions (CDRs) are underlined. SEQ ID NO: VH circuits are shown after the circuit name in the figure (eg, PD1H93_8;_8 indicates SEQ ID NO: 8).

In FIG. 2B for the mAb line PD1B505 shows the alignment of the VL regions of PD1L30 (SEQ ID NO: 14),

- 3 043064

PD1L468 (SEQ ID NO: 15), PD1L469 (SEQ ID NO: 16), PD1L651 (SEQ ID NO: 143) and PD1L652 (SEQ ID

NO: 144). CDRs are underlined. SEQ ID NO: VL circuits are shown after the circuit name in the figure (for example,

PD1L3014; 14 indicates SEQ ID NO: 14).

In FIG. 3A for mAb line PD1B506 shows the alignment of the VH regions of PD1H90 (SEQ ID NO: 44), PD1H388 (SEQ ID NO: 45), PD1H399 (SEQ ID NO: 46), PD1H400 (SEQ ID NO: 47), PD1H401 (SEQ ID NO: 47). NO: 48), PD1H402 (SEQ ID NO: 49), PD1H403 (SEQ ID NO: 50) and PD1H404 (SEQ ID NO: 51). CDRs are underlined.

In FIG. 3B for mAb line PD1B506 shows the alignment of the VL regions of PD1L28 (SEQ ID NO: 60), PD1L470 (SEQ ID NO: 61) and PD1L471 (SEQ ID NO: 62). CDRs are underlined.

In FIG. 4A for mAb line PD1B512 shows the alignment of the VH regions of PD1H81 (SEQ ID NO: 94) and PD1H389 (SEQ ID NO: 95). CDRs are underlined.

In FIG. 4B for mAb line PD1B512 shows the alignment of the VL regions of PD1L43 (SEQ ID NO: 98), PD1L472 (SEQ ID NO: 99) and PD1L473 (SEQ ID NO: 100). CDRs are underlined.

In FIG. 5A shows that PD1B505 and PD1B506 inhibited the activation of antigen-specific T cells. The figure shows the mean % inhibition and std. off T cell proliferation in the CMV secondary response assay. IgG1: isotype control.

In FIG. 5B shows that PD1B743, PD1B750 and PD1B756 inhibited the activation of antigen-specific T cells. The figure shows the mean % inhibition and std. off T cell proliferation in the CMV secondary response assay. IgG1: isotype control.

In FIG. 5C shows that PD1B878 and PD1B849 inhibited the activation of antigen-specific T cells in the CMV specific secondary response assay. The figure shows the mean % inhibition and std. off T cell proliferation in the CMV secondary response assay. IgG1: isotype control.

In FIG. 6A shows that PD1B743 and PD1B756 did not block the binding of PD-L1 to PD-1, while PD1B750 blocked the interaction in the analysis of the degree of clustering of cells expressing PD-1 and PD-L1 in the presence or absence of these antibodies using percentage (% ) double positive events as an indicator of clustering. The positive control mAb blocked the PD-L1/PD-1 interaction.

In FIG. 6B shows that PD1B743 and PD1B756 did not block the binding of PD-L2 to PD-1, while PD1B750 blocked the interaction in the analysis of the degree of clustering of cells expressing PD-1 and PD-L2 in the presence or absence of these antibodies using percentage (% ) double positive events as an indicator of clustering. The positive control mAb blocked the PD-L2/PD-1 interaction.

In FIG. 7 is a diagram of five different epitope groups of the generated anti-PD-1 antibodies. mAbs from group 5 blocked the PD-L1/PD-1 interaction, while mAbs from groups 1-4 did not block it.

In FIG. 8A shows that PD-1 expression was higher on CMV-stimulated CD4 ⁺ CD45RO+ or CD8 ⁺ CD45RO+ memory T cells than on TT-stimulated T cells (inset, geometric mean fluorescence intensity). Anti-PD-1 antibody: solid lines; isotype control: dotted lines.

In FIG. 8B shows that PD1B878 and PD1B849 inhibited the activation of CMV-specific T cells in the CMV specific secondary response assay. The figure shows the average percentage (%) inhibition and stand. off T cell proliferation in this assay.

In FIG. 9A shows that PD1B849 and PD1B878 induced antibody-dependent cell mediated cytotoxicity (ADCC) in activated memory T cells in the presence of NK cells as effector cells. Low fucose (LF) antibody variants (PD1B849-LF and PD1B878-LF) showed improved ADCC activity.

In FIG. 9B shows that PD1B849 and PD1B878 induced ADCC in activated memory T cells in the presence of peripheral blood mononuclear cells (PBMCs) as effector cells. Low fucose (LF) antibody variants (PD1B849-LF and PD1B878-LF) showed improved ADCC activity.

In FIG. 10A shows the absence of PD1B849 and PD1B878 mediated ADCC in resting memory T cells expressing low levels of PD-1 in the presence of NK cells as effector cells. Low fucose (LF) antibody variants (PD1B849-LF and PD1B878-LF) contributed to some ADCC.

In FIG. 10B shows the absence of PD1B849 and PD1B878 mediated ADCC in resting memory T cells expressing low levels of PD-1 in the presence of PBMCs as effector cells. Low fucose (LF) antibody variants (PD1B849-LF and PD1B878-LF) contributed to some ADCC.

In FIG. 11 shows that PD1B849 and PD1B878 did not contribute to measurable complement dependent cytotoxicity (CDC) in activated T cells when rabbit complement was used. OKT3: mouse anti-human CD3 antigen (positive control); huIgG1: isotype control, muIgG2a: isotype control.

- 4 043064

In FIG. 12 shows that PD1B878, PD1B1090 and PD1B1094 did not block PD-L1 binding to PD1 on cells.

In FIG. 13A shows that PD1B505-mIgG2a and PD1B506-mIgG2a prevented disease progression in a graft-versus-host (GvHD) mouse model. Antibodies were administered at a dose of 10 mg/kg ip on days 0, 4, 7, 11, 14 and 18 and clinical scores were recorded over time.

In FIG. 13B shows that mAbs PD1B505-mIgG2a and PD1B506-mIgG2a (mIgG2a) prevented weight loss in a GvHD mouse model. Antibodies were administered at a dose of 10 mg/kg ip on days 0, 4, 7, 11, 14 and 18.

In FIG. 14A shows that PD1B849-mIgG2a and PD1B878-mIgG2a prevented disease progression in a graft-versus-host (GvHD) mouse model. Antibodies were administered at a dose of 10 mg/kg ip on days 0, 4, 7, 11, 14 and 18 and clinical scores were recorded over time.

In FIG. 14B shows that PD1B849-mIgG2a and PD1B878-mIgG2a prevented weight loss in a GvHD mouse model. Antibodies were administered at a dose of 10 mg/kg ip on days 0, 4, 7, 11, 14 and 18.

In FIG. 15 shows that PD1B849-mIgG2a and PD1B878-mIgG2a increased the occurrence of regulatory T cells (Treg) in spleens in a GvHD mouse model.

In FIG. 16 shows that selected anti-PD-1 antibodies deplete T _FH /T _PH cell populations. LF: low fucose.

Detailed description of the invention

All publications, including, without limitation, patents and patent applications cited in this specification, are incorporated herein by reference as if they were set forth herein in their entirety.

It should be understood that the terms used herein are for the purpose of describing embodiments only and are not restrictive. All technical and scientific terms used in this document, unless otherwise indicated, have a generally accepted meaning, understandable to a person of ordinary skill in the field to which the invention pertains.

Exemplary methods and materials are described herein, although any methods and materials similar or equivalent to those described herein may be used in the practice to test the present invention. In describing and presenting the claims of the present invention, the following terms will be used.

Used in the present description and in the claims of the singular include references to multiple objects, unless otherwise follows clearly from the context.

Unless the context clearly implies otherwise, in this description and claims, the words contain, containing, etc. should be interpreted in an inclusive sense, as opposed to an exclusive or exhaustive sense; that is, in the sense of including, without limitation.

The terms specifically binds to or specific binding or binds to refer to the binding of an antibody to an antigen or epitope within the antigen with greater affinity than to other antigens or epitopes. Typically, an antibody binds to an antigen or epitope within the antigen with an equilibrium dissociation constant (KD) of about 1 x 10' ⁷ M or less, such as about 1 x 10'8 M or less, about 1 x 10'9 M or less, about 1 x 10' ¹⁰ M, or less, about 1x10 ^-11 M or less, or about 1x10' ¹² M or less, typically with a KD that is at least one hundred times lower than its KD of binding to a non-specific antigen (eg, BSA, casein). K _D can be measured using standard procedures. However, antibodies that specifically bind to an antigen or epitope within an antigen may be cross-reactive with other related antigens, for example with the same antigen of other species (homologues) such as humans or monkeys, such as Masaca fascicularis (cynomolgus macaque, crabeater), Pan troglodytes (chimpanzee) or Callithrix jacchus (common marmoset, marmoset). Where a monospecific antibody specifically binds one antigen or one epitope, a bispecific antibody specifically binds two different antigens or two different epitopes.

The term agonist or agonist refers to an antibody which, when bound to PD-1, induces at least one PD-L1-inducible biological activity, ie. ligand PD-1. An antibody is an agonist if at least one biological activity is induced by at least about 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% more, than in the absence of an agonist (ie, negative control), or when the induction is statistically significant compared to the induction in the absence of an agonist. A typical biological activity induced by binding of PD-L1 to PD-1 is the inhibition of antigen-specific CD4+ and/or CD8+ T cells, resulting in suppression of immune responses.

PD-1 refers to human programmed cell death protein 1 (PD-1). PD-1 is also known as CD279 or PDCD1. The amino acid sequence of mature human PD-1 (no signal sequence) is shown in SEQ ID NO: 131. The extracellular domain spans residues 1-150, the transmembrane domain spans residues 151-171, and the cytoplasmic

- 5 043064 domain spans residues 172-268 in the sequence of SEQ ID NO: 1. In the text of the description, the extracellular domain of human PD-1 or huPD1-ECD refers to a protein having an amino acid sequence of residues 1-150 in the sequence of SEQ ID NO: 1.

Antibodies are meant in a broad sense and include immunoglobulin molecules belonging to any class, IgA, IgD, IgE, IgG and IgM or subclass, IgA1, IgA ₂ , IgG1, IgG ₂ , IgG ₃ and IgG ₄ , and include light a chain of either kappa (k) or lambda (λ) type. The term antibodies includes monoclonal antibodies, full length antibodies, antigen binding fragments, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies, and any other modified configuration of an immunoglobulin molecule that contains an antigen binding fragment of the desired specificity. Full-length antibodies consist of two heavy chains (HC) and two light chains (LC) connected by disulfide bonds, as well as their multimers (for example, IgM). Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (consisting of CH1 domains, CH2 hinge region and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). VH and VL can be further subdivided into regions of hypervariability, referred to as complementarity determining regions (CDRs), between which are framework regions (FRs). Each VH and VL region consists of three CDRs and four FRs arranged in the amino to carboxy direction in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

Complementarity determining regions (CDRs) are regions of antibodies that bind an antigen. There are three CDRs in the VH region (HCDR1, HCDR2, HCDR3) and three CDRs in the VL region (LCDR1, LCDR2, LCDR3). CDR can be determined using various schemes, for example according to Kabat (Wu et al. (1970) J. Exp. Med. 132: 211-50) (Kabat et al., Sequences of Proteins of Immunological Interest, 5 ^th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al. (1987) J. Mol. Biol. 196: 901-17), IMGT (Lefranc et al. (2003) Dev. Comp. Immunol. 27: 55-77) and AbM (Martin and Thornton J. Bmol. Biol. 263: 800-15, 1996). Correspondence between different schemes and numbering of variable regions is described (see, for example, Lefranc et al. (2003) Dev. Comp. Immunol. 27:5577; Honegger and Pluckthun, J. Mol. Biol. (2001) 309:657-70 ; International ImMunoGeneTics (IMGT) database; web resources, http://www_imgt_org). Available programs such as abYsis from UCL Business PLC can be used to mark up CDRs. As used herein, the designations CDR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 include CDRs defined by any of the methods described above by Kabat, Chothia, IMGT, or AbM, unless otherwise expressly stated herein.

The term antigen-binding fragment refers to the portion of an immunoglobulin molecule that binds an antigen. Antigen-binding fragments may be synthetic, enzymatically produced or genetically engineered polypeptides and include VH, VL, VH and VL, Fab fragments, F(ab') ₂ , Fd and Fv, domain antibodies (dAbs) consisting of from a single VH domain or a single VL domain, shark IgNAR variable domains, camel-adapted VH domains, minimal recognition units consisting of amino acid residues that mimic antibody CDR regions, e.g. FR3-CDR3-FR4, HCDR1, HCDR2 and/or HCDR3, as well as LCDR1, LCDR2 and/or LCDR3. The VH and VL domains can be linked together via a synthetic linker to form various types of single chain antibody configurations, in which the VH/VL domains can be paired intramolecularly or intermolecularly in cases where the VH and VL domains are expressed by separate single chain antibody constructs to form a monovalent antigen-binding site, such as single chain Fv (scFv) or diatelo; they are described, for example, in International Patent Publication Nos. WO 1998/44001, WO 1988/01649, WO 1994/13804 and WO 1992/01047.

The term monoclonal antibody refers to an antibody derived from a substantially homogeneous population of antibody molecules, i. individual antibodies constituting a population that are identical except for possible well-known changes such as removal of the C-terminal lysine from the heavy chain of the antibody, or post-translational modifications such as isomerization or deamidation of amino acids, oxidation of methionine or asparagine, or deamidation of glutamine. Monoclonal antibodies usually bind one antigenic epitope. Bispecific monoclonal antibodies bind to two different antigenic epitopes. Within an antibody population, monoclonal antibodies may have heterogeneous glycosylation. A monoclonal antibody can be monospecific or multispecific, such as bispecific, as well as monovalent, divalent or multivalent.

The term isolated refers to a homogeneous population of molecules (such as synthetic polynucleotides or a protein such as an antibody) that have been substantially separated and/or purified from other components of the system in which the molecules were formed, such as a recombinant cell, as well as a protein , which has been subjected to at least one purification or isolation step. The term isolated antibody refers to an antibody that is substantially free of other cellular

- 6 043064 materials and / or chemicals, and covers antibodies that are isolated with relatively high purity, such as 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 , 94, 95, 96, 97, 98, 99 or 100% pure.

The term antibodies includes antibodies obtained using various technologies, including antibodies obtained from immunized animals such as mice, rats, rabbits or chickens, or identified from phage display libraries or mammalian displays, as described herein.

The term humanized antibody refers to an antibody in which at least one CDR is derived from a non-human species and at least one framework is derived from human immunoglobulin sequences. The humanized antibody may include substitutions in the framework regions, whereby the frameworks may not be an exact copy of the expressed human immunoglobulin or human germline gene sequences.

The term human antibody refers to an antibody that is optimized to provide a minimal immune response when administered to a human individual. The variable regions of a human antibody are derived from human germline immunoglobulin sequences. If the antibody contains a constant region or a portion of a constant region, then the constant region is also derived from human germline immunoglobulin sequences.

A human antibody contains heavy or light chain variable regions that are derived from human germline immunoglobulin sequences when the antibody variable regions are derived from a system that uses human germline immunoglobulin genes. Examples of such systems include human immunoglobulin gene libraries displayed on phages or mammalian cells, and non-human transgenic animals such as mice, rats or chickens carrying human immunoglobulin loci. A human antibody typically contains amino acid differences compared to immunoglobulins expressed in humans due to differences between the systems used to generate antibodies and human immunoglobulin loci, the introduction of naturally occurring somatic mutations, the intentional introduction of substitutions in framework regions or CDRs. Typically, a human antibody is at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 % identical to the amino acid sequence encoded by the human germline immunoglobulin sequence. In some instances, the human antibody may contain consensus framework sequences derived from human framework sequence assays, for example, as described in Knappik et al. (2000) J. Mol. Biol. 296: 57-86), or synthetic HCDR3 included in human immunoglobulin gene libraries displayed on phage, for example, as described in (Shi et al. (2010) J. Mol. Biol. 397: 385-96) and in the international Patent Publication No. WO 2009/085462. Antibodies in which the CDRs are derived from a non-human species do not fit the definition of a human antibody.

The term recombinant refers to antibodies and other proteins that are produced, expressed, generated or isolated by recombinant means.

The term epitope refers to the portion of an antigen to which an antibody specifically binds. Typically, epitopes are composed of reactive (such as polar, nonpolar, or hydrophobic) surface groupings of components such as amino acid or polysaccharide side chains, and may have specific three-dimensional structural characteristics as well as specific charge characteristics. An epitope may be formed from continuous and/or discontinuous amino acids forming a conformational spatial unit. In the case of a discontinuous epitope, amino acids from different parts of the linear sequence of the antigen come close to each other in 3-dimensional space by folding the protein molecule.

The term multispecific refers to a protein, such as an antibody, that specifically binds to two or more different antigens or two or more different epitopes within the same antigen. The multispecific protein may be cross-reactive with other related antigens, for example the same antigen from other species (homologs) such as humans or monkeys, e.g. Masaca fascicularis (cynomolgus macaque), Pan troglodytes (chimpanzee), Callithrix jacchus (common marmoset , marmoset), or may bind an epitope that is present on two or more different antigens.

The term bispecific refers to a protein, such as an antibody, that specifically binds to two different antigens or two different epitopes within the same antigen. The bispecific protein may be cross-reactive with other related antigens, such as the same antigen from other species (homologs) such as humans or monkeys, e.g. Masaca fascicularis (cynomolgus macaque), Pan troglodytes (chimpanzee), Callithrix jacchus (common marmoset , marmoset), or may bind an epitope that is present on two or more different antigens.

The expression in combination with means that drugs or therapeutic agents are administered to a patient, such as a human, together in admixture, simultaneously as separate agents, or sequentially as separate agents in any order.

- 7 043064

The terms "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventive measures, the aim being to prevent or slow down (reduce) an undesired physiological change or disorder. Beneficial or desirable clinical outcomes include relief of symptoms, reduction in the extent of the disease, stabilization (i.e. no worsening) of the course of the disease, delay or retardation of the progression of the disease, alleviation or temporary amelioration of the disease state, and remission (partial or complete) as detectable, so undetectable. The term treatment can also mean prolongation of survival compared to that expected in the absence of a patient's treatment. Patients in need of treatment include those who already have the condition or disorder, as well as those who are predisposed to developing the condition or disorder, or those in whom such condition or disorder is to be prevented.

The term therapeutically effective amount refers to an amount effective at the dosages and for the periods of time necessary to achieve the desired therapeutic result. The therapeutically effective amount may vary depending on such factors as the course of the disease, the age, sex and body weight of the subject, as well as the ability of the therapeutic agent or combination of therapeutic agents to elicit the desired response in the subject. Examples of features of an effective therapeutic agent or combination of therapeutic agents include, for example, improving the health of the patient.

The term immune response includes immune responses mediated by T cells and/or B cells. Examples of immune responses include T cell responses such as cytokine production and cell mediated cytotoxicity. In addition, the term immune response includes immune responses that are indirectly affected by T cell activation, such as antibody production (humoral responses), and activation of cytokine-responsive cells, such as macrophages.

The term reduce or reduce refers to a measurable decrease in the level of immune response in a patient compared to the level of response in a patient in the absence of treatment or compound, and/or compared to the level of response in an otherwise identical but untreated patient.

The term immune disorder refers to any disease, disorder, or disease symptom caused by the activity of the immune system, including autoimmune diseases, inflammatory diseases, and allergic reactions.

The term patient includes any human or non-human animal. The term non-human animal includes all vertebrates, such as mammals and non-mammals such as primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. The terms subject and patient may be used herein interchangeably.

The term vector refers to a polynucleotide capable of duplication within a biological system or which can be moved between such systems. Vector polynucleotides typically contain elements such as origins of replication, a polyadenylation signal, or selectable markers, the function of which is to facilitate duplication or retention of such polynucleotides in a biological system. Examples of such biological systems may include cell, virus, animal, plant, and reengineered biological systems using biological components capable of vector doubling. The vector-containing polynucleotide may be a DNA or RNA molecule or a hybrid thereof.

The term expression vector means a vector that can be used in a biological system or a reshaped biological system to direct translation of a polypeptide encoded by a polynucleotide sequence present in an expression vector.

The term polynucleotide refers to a synthetic molecule containing a chain of nucleotides covalently linked through a sugar phosphate backbone or other equivalent covalent chemical structure, DNA is an example of a synthetic polynucleotide.

The term polypeptide or protein refers to a molecule that contains at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides containing less than 50 amino acid residues may be referred to as peptides.

The term about means within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, the error depending in part on how that value is measured or determined, ie. from the limitations of the measurement system. Unless explicitly stated otherwise in the examples or elsewhere herein in the context of a particular assay, result, or embodiment, the term about means within one standard deviation, in accordance with practice accepted in the art, or up to 5%, in whichever is greater.

The term sample refers to a collection of similar fluids, cells, or tissues isolated from a patient's body, as well as fluids, cells, or tissues within a patient. Examples of samples are biological fluids such as blood, serum and serous fluids, plasma, lymph, urine, saliva, cystic fluid, tears, feces, sputum, mucus

- 8 043064 secretory tissues and organs, vaginal discharge, ascitic fluid, fluids in the pleura, pericardium, peritoneum, abdominal and other body cavities, fluids collected by flushing from the bronchi, synovial fluid, liquid solutions in contact with the patient or biological source, such as cell and organ culture media, including conditioned cell and organ media, washes, and the like, tissue biopsy specimens, fine needle aspirates, surgically resected tissue, organ cultures, or cell cultures.

In this document, conventional one- and three-letter codes for designating amino acids are used, as shown in table. 1.

Table 1

Aminokis- lot Three letter code Single letter code Aminokis- lot Three letter code Single letter code Alanine Ala A Leucine Leu L Arginine Arg R Lysine Lys TO Asparagine Asn N Methionine Met M Aspartate asp D Phenylalanine Phe F Cysteine Cys C Proline Pro P Glutamate gin E Serene Ser S Glutamine Glu Q Threonine Thr T Glycine gly G tryptophan trp w G istidine His H Tyrosine Tyr Y Isoleucine He I Valine Vai V

Compositions of the invention.

Provided herein are antibodies that specifically bind to PD-1 or antigen-binding fragments thereof, polynucleotides encoding the antibodies provided herein, vectors, host cells, and methods for making and using antibodies. The antibodies, or antigen-binding fragments thereof, may be agonistic antibodies.

Some cancer patients treated with immune checkpoint inhibitors, including PD-1 antagonists, develop autoimmune adverse events such as symptoms of arthritis, colitis, or psoriasis. One hypothesis to explain this observation is that autoreactive T cells in these patients were actively downregulated via PD-1 and released in the presence of a PD-1 antagonist. Subsequently, it is worth looking at this hypothesis from a different angle, and consider it likely that already released T cells in patients who have an autoimmune disease can be suppressed through PD-1 ligation/agonism.

Single nucleotide polymorphisms (SNPs) in the PD-1 gene, PDCD1, have been found to be associated with various autoimmune diseases, including rheumatoid arthritis, lupus, and ankylosing spondylitis (reviewed in Zamani et al., Cell Immunol. 2016 310:27-41) . Although the functions of SNPs in PD-1 have not yet been identified, these associations may indicate that a decrease in PD-1 activity may lead to a decrease in T cell suppression, which may increase susceptibility to autoimmune disease.

There is a need for therapeutic agents to suppress autoimmune T cells in autoimmune diseases. PD-1+ T cells have been found in tissues of patients with autoimmune diseases, including rheumatoid arthritis and Sjögren's syndrome (Wan et al., J. Immunol. 2006 177(12):8844-50; Kobayashi et al., J. Rheumatol 2005 32(11):2156-63). An antibody capable of agonizing against PD-1 can be used to suppress T cell proliferation and release of cytokines, to limit tissue damage, and to restore immune homeostasis. PD-1 mAb agonists will target activated rather than resting intact T cells and memory T and B cells. Thus, the therapeutic agent will suppress immune responses to autoantigens in patients with autoimmune diseases without compromising immune memory responses to pathogens. Two types of T cells that express high levels of PD-1, TFH and TPH stimulate B cell responses and antibody production (Rao et al., Nature 2017; 542: 110-114). The frequency of these cells is increased in autoimmune diseases caused by the production of autoantibodies, including rheumatoid arthritis, systemic lupus erythematosus and Sjögren's syndrome (Rao et al., Nature 2017; 542: 110-114; He et al., Immunity 2013; 39: 770- 781; Verstappen et al., Arthr & Rheum 2017; 69(9): 1850-1861). The antibodies of the invention, in addition to providing suppression of activated T cells, can selectively kill cells highly expressing PD-1, such as TFH and _TPH cells.

The invention provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof.

- 9 043064

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3, a light chain complementarity determining region 1 (LCDR1), LCDR2 and/or LCDR3 any of the antibodies PD1B505, PD1B742, PD1B743, PD1B878, PD1B506, PD1B750, PD1B751, PD1B845, PD1B846, PD1B847, PD1B848, PD1B849, PD1B850, PD1B512, PD1B756 , PD1B757, PD1B1085, PD1B1086, PD1B1087, PD1B1088, PD1B1089, PD1B1090, PD1B1091, PD1B1092 , PD1B1093, PD1B1094 or PD1B1095.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising a heavy chain variable region (VH) framework and/or a light chain variable region framework (VL) of any of the antibodies PD1B505, PD1B742, PD1B743, PD1B878 ,PD1B506,PD1B750,PD1B751,PD1B845,PD1B846,PD1B847,PD1B848,PD1B849,PD1B850,PD1B512,PD1B756,PD1B757,PD1B1085,PD1B1086,PD1B10 87, PD1B1088, PD1B1089, PD1B1090, PD1B1091, PD1B1092, PD1B1093, PD1B1094 or PD1B1095.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, containing the VH and/or VL of any of the antibodies PD1B505, PD1B742, PD1B743, PD1B878, PD1B506, PD1B750, PD1B751, PD1B845, PD1B846, PD1B847, PD1B848 PD1B1087 , PD1B1094 or PD1B1095.

In some embodiments, the antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, is an agonist antibody.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, mediates ADCC in cells expressing PD-1.

In some embodiments, the cells expressing PD-1 are activated memory T cells, follicular helper T cells (T _FH ), peripheral helper T cells (T _PH ), or any combination thereof.

T _FH cells can be identified as: live CD19-CD567CD4+CD45RO+/HLADR+/CXCR5+/ ICOS+PD1+;

_TPH cells can be identified as: live CD19-CD567CD4+CD45RO+/HLADR+/CXCR57 ICOS+PD1+;

combination of T _FH /T _PH cells can be identified as: live CD19'CD56'/CD4 ⁺ CD45RO ⁺ / HLADR7ICOS+PD17

Memory T cells can be identified as CD4 ⁺ CD45RO+ or CD8 ⁺ CD45RO+.

Antibodies of the PD1B505 line.

mAbs such as PD1B505, PD1B742, PD1B743, PD1B878, PD1B1085, PD1B1086, PD1B1087, PD1B1088, PD1B1089, PD1B1090, PD1B1091, PD1B1092, PD1B1093, PD1 B1O94 or PD1B1O95 are examples of mAb PD1B505 antibodies. These mAbs have identical CDR regions, except that some antibodies have a one amino acid difference in HCDR2 and a one or two amino acid difference in LCDR1. The identity of the VH region is in the range of 82-100%, and the identity of the VL region is in the range of 78-100%. mAbs of the PD1B505 line are non-ligand blocking.

The line is characterized by HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 2, 165, 4, 166, 6 and 7, respectively, and the VH genomic sequence with SEQ ID NO: 118, and the VL genomic sequence with SEQ ID NO : 119.

SEQ ID NO: 165 (genomic HCDR2) WINIETGXPT; and

X is E, Y, H or W.

SEQ ID NO: 166 (genomic LCDR1) TASSSX1X2SSYLH; and

X1 is V or F; And

X2 is S or P.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOS: 2, 165, 4, 166, 6 and 7, respectively.

In some embodiments, an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprises HCDR2 of SEQ ID NO: 3, 145, 146, or 147 and/or LCDR1 of SEQ ID NO: 5, 148, or 149.

In some embodiments, the antibody, or antigen-binding fragment thereof, has one, two, three, four, or five of the following properties:

does not block the binding of PD-L1 to PD-1, and the absence of blocking is measured by the inability of the antibody to inhibit the clustering of cells expressing PD-L1 and cells expressing PD-1, as described in example 1;

binds to PD-1 with an equilibrium dissociation constant (KD) of about 5x10'8 M or less, with KD measured using the ProteOn XPR36 system at +25°C;

- 10 043064 binds to PD-1 with an association constant (Ka) of about 3x10 ⁴ 1/Ms or more, and Ka is measured using the ProteOn XPR36 system at +25°C;

binds to PD-1 with a dissociation constant (Kd) of about 3x10 ³ 1/s or less, and Kd is measured using the ProteOn XPR36 system at +25°C; or inhibits the proliferation of antigen-specific T cells; while proliferation is assessed in the CMV-PBMC assay.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprises a VH framework derived from IGHV7-4-1*1 (SEQ ID NO: 125).

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprises a VL framework derived from IGKV3D-20*1 (SEQ ID NO: 126).

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprises a VH framework derived from IGHV7-4-1*1 (SEQ ID NO: 125) and a VL framework derived from IGKV3D-20*1 ( SEQ ID NO: 126).

SEQ ID NO: 125 IGHV7-4-l*l

QVQLVQSGSELKKPGASVKVSCKASGYTFTSYAMNWVRQAPGQGLEWMGWIN

TNTGNPTYAQGFTGRFVFSLDTSVSTAYLQICSLKAEDTAVYYCAR

SEQ ID NO: 126 IGKV3D-20*l

EIVLTQSPATLSLSPGERATLSCGASQSVSSSYLAWYQQKPGLAPRLLIYDASSRA

TGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising the heavy chain variable region (VH) of SEQ ID NO: 118. SEQ ID NO: 118 is the VH genomic sequence from mAb line PD1B505.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising the light chain variable region (VL) of SEQ ID NO: 119. SEQ ID NO: 118 is the VL genomic sequence from mAb line PD1B505.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising VH of SEQ ID NO: 118 and VL of SEQ ID NO: 119. CDRs are shown in bold in SEQ ID NO: 118 and SEQ ID NO: 119.

SEQ ID NO: 118 (genomic VH lineage PD1B505)

XiVQLX2X3SGX4ELKKPGX ₅ X6VKX ₇ SCKASGYTFTDYSMHWVX ₈ QAPGX ₉ GLXio

WMGWINIETGX11PTYAX12X13FX14GRFX15FSLX16TSX17STAYLQIX18X19LKX20EDTAX21

YFCARDYYGTYFYAMDYWGQGTX22X23TVSS;

and

X1 is D or Q;

X ₂ is Q or V;

X ₃ represents E or Q;

X ₄ is P or S;

X ₅ represents E or A;

X6 is T or S;

X ₇ represents I or V;

X8 is K or R;

X9 is K or Q;

X ₁₀ is K or E;

X11 is E, Y, H or W;

X ₁₂ is D or Q;

X ₁₃ is D or G;

X ₁₄ is K or T;

X ₁₅ is A or V;

X ₁₆ is E or D;

X ₁₇ is A or V;

X ₁₈ is N, C or S;

X ₁₉ is N or S;

X ₂₀ is N or A;

X ₂₁ is T or V;

X ₂₂ is T or L; or

X ₂₃ is L or V.

SEQ ID NO: 119 (genomic VL line PD1B505)

- 11 043064

XiIYLTQSPAX2X3SX4SX ₅ GERX6TX ₇ X ₈ CTASSSX9XioSSYLHWYQQKPGXiiXi ₂ PXi 3LXi4lYSTSNLASGXi ₅ PXi6RFSGSGSGTXi ₇ Xi ₈ Xi9LTISX2oX2iEX22EDX23AX2 ₄ YYCH QYHRSPLTFGX2 ₅ GTK LEX2 _6K ;

and

X ₁ is Q or E;

X ₂ represents I or T;

X ₃ is M or L;

X ₄ is A or L;

X ₅ is L or P;

X6 is V or A;

X ₇ is M or L;

X ₈ is T or S;

X ₉ is V or F;

X ₁₀ is S or P;

X _n is S or L;

X ₁₂ is S or A;

X ₁₃ is K or R;

X ₁₄ is W or L;

X ₁₅ is V or I;

X ₁₆ is A or D;

X ₁₇ is S or D;

X ₁₈ is Y or F;

X ₁₉ is S or T;

X ₂₀ is S or R;

X ₂₁ is M or L;

X ₂₂ is A or P;

X23 is A or F;

X ₂₄ is T or V;

X ₂₅ is A or Q; And

X ₂₆ is L or I.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprises a VH of SEQ ID NOS: 8, 9, 10, 140, 141, or 142.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprises a VL of SEQ ID NO: 14, 15, 16, 143, or 144.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprises a VH of SEQ ID NOs: 8, 9, 10, 140, 141, or 142 and a VL of SEQ ID NOs: 14, 15, 16, 143 or 144.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOS: 2, 3, 4, 5, 6 and 7, respectively.

In some embodiments, the antibody comprises a VH of SEQ ID NO: 8, 9, or 10 and a VL of SEQ ID NO: 14, 15, or 16.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 20, 21, or 22 and a light chain (LC) of SEQ ID NO: 26, 27, or 28.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOS: 2, 145, 4, 5, 6 and 7, respectively. In some embodiments, the antibody comprises VH of SEQ ID NO: 140 and VL of SEQ ID NO: 16. In some embodiments, the antibody comprises HC of SEQ ID NO: 150 and LC of SEQ ID NO: 28. Also provided is an antibody for use in therapy, for example in the treatment of an immune disorder, rheumatoid arthritis, lupus, systemic lupus erythematosus, or graft versus host disease.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOS: 2, 146, 4, 5, 6 and 7, respectively. In some embodiments, the antibody comprises VH of SEQ ID NO: 141 and VL of SEQ ID NO: 16. In some embodiments, the antibody comprises HC of SEQ ID NO: 151 and LC of SEQ ID NO: 28. Also provided is an antibody for use in therapy, for example in the treatment of an immune disorder, rheumatoid arthritis, lupus, systemic lupus erythematosus, or graft versus host disease.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOS: 2, 147, 4, 5, 6 and 7, respectively. In some embodiments, the antibody

- 12 043064 contains VH with SEQ ID NO: 142 and VL with SEQ ID NO: 16. In some embodiments, the antibody contains HC with SEQ ID NO: 152 and LC with SEQ ID NO: 28. An antibody is also proposed for use in therapy, for example, in the treatment of an immune disorder, rheumatoid arthritis, lupus, systemic lupus erythematosus, or graft versus host disease.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOS: 2, 3, 4, 148, 6 and 7, respectively. In some embodiments, the antibody comprises VH of SEQ ID NO: 10 and VL of SEQ ID NO: 143. In some embodiments, the antibody comprises HC of SEQ ID NO: 22 and LC of SEQ ID NO: 153. Also provided is an antibody for use in therapy, for example in the treatment of an immune disorder, rheumatoid arthritis, lupus, systemic lupus erythematosus, or graft versus host disease.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOS: 2, 3, 4, 149, 6 and 7, respectively. In some embodiments, the antibody comprises VH of SEQ ID NO: 10 and VL of SEQ ID NO: 144. In some embodiments, the antibody comprises HC of SEQ ID NO: 22 and LC of SEQ ID NO: 154. Also provided is an antibody for use in therapy, for example in the treatment of an immune disorder, rheumatoid arthritis, lupus, systemic lupus erythematosus, or graft versus host disease.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOS: 2, 145, 4, 148, 6 and 7, respectively. In some embodiments, the antibody comprises VH of SEQ ID NO: 140 and VL of SEQ ID NO: 143. In some embodiments, the antibody comprises HC of SEQ ID NO: 150 and LC of SEQ ID NO: 153. Also provided is an antibody for use in therapy, for example in the treatment of an immune disorder, rheumatoid arthritis, lupus, systemic lupus erythematosus, or graft versus host disease.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOS: 2, 146, 4, 148, 6 and 7, respectively. In some embodiments, the antibody comprises VH of SEQ ID NO: 141 and VL of SEQ ID NO: 143. In some embodiments, the antibody comprises HC of SEQ ID NO: 151 and LC of SEQ ID NO: 153. Also provided is an antibody for use in therapy, for example in the treatment of an immune disorder, rheumatoid arthritis, lupus, systemic lupus erythematosus, or graft versus host disease.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOS: 2, 147, 4, 148, 6 and 7, respectively. In some embodiments, the antibody comprises VH of SEQ ID NO: 142 and VL of SEQ ID NO: 143. In some embodiments, the antibody comprises HC of SEQ ID NO: 152 and LC of SEQ ID NO: 153. Also provided is an antibody for use in therapy, for example in the treatment of an immune disorder, rheumatoid arthritis, lupus, systemic lupus erythematosus, or graft versus host disease.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOS: 2, 145, 4, 149, 6 and 7, respectively. In some embodiments, the antibody comprises VH of SEQ ID NO: 140 and VL of SEQ ID NO: 144. In some embodiments, the antibody comprises HC of SEQ ID NO: 150 and LC of SEQ ID NO: 154. Also provided is an antibody for use in therapy, for example in the treatment of an immune disorder, rheumatoid arthritis, lupus, systemic lupus erythematosus, or graft versus host disease.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOS: 2, 146, 4, 149, 6 and 7, respectively. In some embodiments, the antibody comprises VH of SEQ ID NO: 141 and VL of SEQ ID NO: 144. In some embodiments, the antibody comprises HC of SEQ ID NO: 151 and LC of SEQ ID NO: 154. Also provided is an antibody for use in therapy, for example in the treatment of an immune disorder, rheumatoid arthritis, lupus, systemic lupus erythematosus, or graft versus host disease.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOS: 2, 147, 4, 149, 6 and 7, respectively. In some embodiments, the antibody comprises VH of SEQ ID NO: 142 and VL of SEQ ID NO: 144. In some embodiments, the antibody comprises HC of SEQ ID NO: 152 and LC of SEQ ID NO: 154. Also provided is an antibody for use in therapy, for example in the treatment of an immune disorder, rheumatoid arthritis, lupus, systemic lupus erythematosus, or graft versus host disease.

In some embodiments, the antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, is an agonist antibody.

- 13 043064

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, wherein the antibody competes for PD-1 binding with an antibody or antigen-binding fragment containing

VH with SEQ ID NO 8 and VL c SEQ ID NO: 14; VH with SEQ ID NO 9 and VL c SEQ ID NO: 15; VH with SEQ ID NO 9 and VL c SEQ ID NO: 16; VH with SEQ ID NO 10 and VL c SEQ ID NO: 16; VH with SEQ ID NO 140 and VL c SEQ ID NO: 16; VH with SEQ ID NO 141 and VLc SEQ ID NO: 16; VH with SEQ ID NO 142 and VL c SEQ ID NO: 16; VH with SEQ ID NO 10 and VL c SEQ ID NO: 143; VH with SEQ ID NO 10 and VL c SEQ ID NO: 144; VH with SEQ ID NO 140 and VL c SEQ ID NO: 143; VH with SEQ ID NO 141 and VLc SEQ ID NO: 143; VH with SEQ ID NO 142 and VL c SEQ ID NO: 143; VH with SEQ ID NO 140 and VL c SEQ ID NO: 144; VH with SEQ ID NO 141 and VL c SEQ ID NO: 144; or VH with SEQ ID NO 142 and VL c SEQ ID NO: 144.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, wherein the antibody binds to the same epitope that binds to an antibody containing

VH with SEQ ID NO: 8 and VL with SEQ ID NO: 14;

VH c SEQ ID NO: 9 and VL c SEQ ID NO: 15;

VH c SEQ ID NO: 9 and VL c SEQ ID NO: 16;

VH c SEQ ID NO: 10 and VL c SEQ ID NO: 16;

VH c SEQ ID NO: 140 and VL c SEQ ID NO: 16;

VH c SEQ ID NO: 141 and VL c SEQ ID NO: 16;

VH c SEQ ID NO: 142 and VL c SEQ ID NO: 16;

VH c SEQ ID NO: 10 and VL c SEQ ID NO: 143;

VH c SEQ ID NO: 10 and VL c SEQ ID NO: 144;

VH c SEQ ID NO: 140 and VL c SEQ ID NO: 143;

VH c SEQ ID NO: 141 and VL c SEQ ID NO: 143;

VH c SEQ ID NO: 142 and VL c SEQ ID NO: 143;

VH c SEQ ID NO: 140 and VL c SEQ ID NO: 144;

VH c SEQ ID NO: 141 and VL c SEQ ID NO: 144; or

VH c SEQ ID NO: 142 and VL c SEQ ID NO: 144.

In some embodiments, the VH and VL regions or the HC and LC regions of an antibody that specifically binds to PD-1 or an antigen-binding fragment provided herein are encoded by a polynucleotide containing a polynucleotide sequence with

SEQ ID NOs: 11 and 17, respectively;

SEQ ID NO: 12 and 18, respectively;

SEQ ID NO: 12 and 19, respectively;

SEQ ID NO: 13 and 19, respectively;

SEQ ID NO: 23 and 29, respectively;

SEQ ID NO: 24 and 30, respectively;

SEQ ID NOs: 24 and 31, respectively;

SEQ ID NOs: 25 and 31, respectively;

SEQ ID NOs: 132 and 133, respectively; or

SEQ ID NO: 134 and 135, respectively;

SEQ ID NOs: 155 and 19, respectively;

SEQ ID NOs: 156 and 19, respectively;

SEQ ID NOs: 157 and 19, respectively;

SEQ ID NO: 13 and 158, respectively;

SEQ ID NO: 13 and 159, respectively;

SEQ ID NO: 155 and 158, respectively;

SEQ ID NO: 156 and 158, respectively;

SEQ ID NO: 157 and 158, respectively;

- 14 043064

SEQ ID NO: 155 and 159, respectively;

SEQ ID NO: 156 and 159, respectively;

SEQ ID NOs: 157 and 159, respectively;

SEQ ID NOs: 160 and 31, respectively;

SEQ ID NOs: 161 and 31, respectively;

SEQ ID NOs: 162 and 31, respectively;

SEQ ID NO: 25 and 163, respectively;

SEQ ID NO: 25 and 164, respectively;

SEQ ID NOs: 160 and 163, respectively;

SEQ ID NO: 161 and 163, respectively;

SEQ ID NO: 162 and 163, respectively;

SEQ ID NO: 160 and 164, respectively;

SEQ ID NO: 161 and 164, respectively; or

SEQ ID NO: 162 and 164, respectively.

Antibodies of the PD1B506 line.

mAbs such as PD1B506, PD1B750, PD1B751, PD1B845, PD1B846, PD1B847, PD1B848, PD1B849, and PD1B850 are examples of the PD1B506 mAb lineage. These mAbs have identical HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3 and HCDR2 variant. The identity of the VH region is in the range of 80-100%, and the identity of the VL region is about 98%. mAbs of the PD1B506 line are ligand-blocking. The line is characterized by HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 32, 124, 40, 41, 42 and 43, respectively, and the VH genomic sequence with SEQ ID NO: 120, and the VL genomic sequence with SEQ ID NO : 121.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2 and HCDR3 of SEQ ID NOS: 32, 124, 40, 41, 42 and 43, respectively.

SEQ ID NO: 124 (genomic HCDR2 lineage PD1B506)

and

X ₁ represents N, D, Q, K or E; And

X ₂ is G, A or I.

In some embodiments, an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprises HCDR2 of SEQ ID NOS: 33, 34, 35, 36, 37, 38, or 39.

In some embodiments, an antibody or antigen-binding fragment provided herein has one, two, three, four, or five of the following properties:

blocks the binding of PD-L1 to PD-1, and blocking is measured by the ability of the antibody to inhibit the clustering of cells expressing PD-L1 and cells expressing PD-1, as described in example 1;

binds to PD-1 with an equilibrium dissociation constant (K _D ) of about 5x10 ^-8 M or less, and K _D is measured using the ProteOn XPR36 system at +25°C;

binds to PD-1 with an association constant (Ka) of about 4x10 ⁵ 1/Ms or more, and Ka is measured using the ProteOn XPR36 system at +25°C;

binds to PD-1 with a dissociation constant (Kd) of about 1 x 10 ^-2 1/s or less, and Kd is measured using the ProteOn XPR36 system at +25°C; or inhibits the proliferation of antigen-specific T cells; while proliferation is assessed in the CMV-PBMC assay as described in Example 1.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprises a VH framework derived from IGHV1-2*02 (SEQ ID NO: 127).

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprises a VL framework derived from IGKV1D-16*1 (SEQ ID NO: 128).

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprises a VH framework derived from IGHV1-2*02 (SEQ ID NO: 127) and a VL framework derived from IGKV1D-16*1 IG (SEQ ID NO: 128).

SEQ ID NO: 127 IGHV 1-2*02

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWI

NPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR

SEQ ID NO: 128 IGKV1D-16*1

DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQS

GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYP

The invention also provides an isolated antibody that specifically binds to PD-1,

- 15 043064 or its antigen-binding fragment containing the variable region of the heavy chain (VH) with SEQ ID

NO: 120. SEQ ID NO: 120 is the VH genomic sequence from mAb line PD1B506.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising a light chain variable region (VL) of SEQ ID

NO: 121. SEQ ID NO: 121 is the VL genomic sequence from mAb line PD1B506.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising VH of SEQ ID NO: 120 and VL of SEQ ID NO: 121. CDRs are shown in bold in SEQ ID NO: 120 and SEQ ID NO: 121.

SEQ ID NO: 120 (genomic VH line PD1B506)

QVQLXiQX ₂ GAEX3X4KPGASVKX ₅ SCKASGYTFTTYWMHWVX ₆ QX7PGQGLEW X ₈ GEINPNX ₉ XioGINY X11X12KFX13X14X15X16TLTVDKSX17STAYMX1 ₈ LSX1 ₉ LX2OSX21D X22AVYYCTIDYYDYGGYWG QGTX23X24TVSS; wherein X ₁ is Q or V;

X ₂ is S or P;

X ₃ is L or V;

X ₄ is V or K;

X ₅ is L or V;

X ₆ is K or R;

X ₇ is R or A;

X ₈ is I or M;

X ₉ is N, D, Q, K or E;

X10 is G, A or I;

X11 is N or A;

X ₁₂ is E or Q;

X13 is K or Q;

X ₁₄ is K or G;

X15 is K or R;

X ₁₆ is A or V;

X ₁₇ is S or I;

X18 is Q or E;

X ₁₉ is S or R;

X ₂₀ is T or R;

X ₂₁ represents E or D;

X ₂₂ is S or T;

X ₂₃ is T or L; And

X24 is L or V.

SEQ ID NO: 121 (genomic VL line PD1B506)

DIX ₁ MTQSX ₂ X ₃ X ₄ X5SX6SVX ₇ DRVX ₈ X ₉ TCKASQNVGTNVAWYQQKPXioXiiXi ₂ P KXi3LIYSASYRYSGVPXi4RFXi ₅ GSGSGTDFTLTIXi ₆ Xi ₇ X ₁₈ QX ₁₉ EDX2oAX ₂ iYX22CQQYNI YPYTFGX _{23GTKLEX 24K} ; wherein X ₁ is V or Q;

X ₂ is Q or P;

X3 is K or S;

X ₄ is F or S;

X ₅ is M or L;

X ₆ is T or A;

X ₇ is R or G;

X ₈ is S or T;

X ₉ is V or I;

X ₁₀ is G or E;

X11 is Q or K;

X ₁₂ is S or A;

X ₁₃ is A or S;

X ₁₄ is D or S;

X ₁₅ is T or S;

X ₁₆ is T or S;

X ₁₇ is N or S;

X ₁₈ is V or L;

- 16 043064

X ₁₉ is S or P;

X2o is L or F;

X ₂₁ represents E or T;

X ₂₂ is F or Y;

X ₂₃ is S or Q; And

X ₂₄ is M or I.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprises a VH of SEQ ID NOS: 44, 45, 46, 47, 48, 49, 50, or 51.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprises a VL of SEQ ID NO: 60, 61, or 62.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprises a VH of SEQ ID NOs: 44, 45, 46, 47, 48, 49, 50, or 51 and a VL of SEQ ID NOs: 60, 61 or 62.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOS: 32, 33, 40, 41, 42 and 43, respectively. In some embodiments, the antibody comprises a VH of SEQ ID NO: 44 or 45 and a VL of SEQ ID NO: 60, 61, or 62. In some embodiments, the antibody comprises an HC of SEQ ID NO: 66 or 67 and an LC of SEQ ID NO: 82, 83, or 84. Also provided is an antibody for use in therapy, for example, in the treatment of an immune disorder, rheumatoid arthritis, lupus, systemic lupus erythematosus, or graft versus host disease.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOS: 32, 34, 40, 41, 42 and 43, respectively. In some embodiments, the antibody comprises VH of SEQ ID NO: 46 and VL of SEQ ID NO: 61. In some embodiments, the antibody comprises HC of SEQ ID NO: 68 and LC of SEQ ID NO: 83. Also provided is an antibody for use in therapy, for example in the treatment of an immune disorder, rheumatoid arthritis, lupus, systemic lupus erythematosus, or graft versus host disease.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOS: 32, 35, 40, 41, 42 and 43, respectively. In some embodiments, the antibody comprises VH of SEQ ID NO: 47 and VL of SEQ ID NO: 61. In some embodiments, the antibody comprises HC of SEQ ID NO: 69 and LC of SEQ ID NO: 83. Also provided is an antibody for use in therapy, for example in the treatment of an immune disorder, rheumatoid arthritis, lupus, systemic lupus erythematosus, or graft versus host disease.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOS: 32, 36, 40, 41, 42 and 43, respectively. In some embodiments, the antibody comprises VH of SEQ ID NO: 48 and VL of SEQ ID NO: 61. In some embodiments, the antibody comprises HC of SEQ ID NO: 70 and LC of SEQ ID NO: 83. Also provided is an antibody for use in therapy, for example in the treatment of an immune disorder, rheumatoid arthritis, lupus, systemic lupus erythematosus, or graft versus host disease.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOS: 32, 37, 40, 41, 42 and 43, respectively. In some embodiments, the antibody comprises VH of SEQ ID NO: 49 and VL of SEQ ID NO: 61. In some embodiments, the antibody comprises HC of SEQ ID NO: 71 and LC of SEQ ID NO: 83. Also provided is an antibody for use in therapy, for example in the treatment of an immune disorder, rheumatoid arthritis, lupus, systemic lupus erythematosus, or graft versus host disease.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOS: 32, 38, 40, 41, 42 and 43, respectively. In some embodiments, the antibody comprises VH of SEQ ID NO: 50 and VL of SEQ ID NO: 61. In some embodiments, the antibody comprises HC of SEQ ID NO: 72 and LC of SEQ ID NO: 83. Also provided is an antibody for use in therapy, for example in the treatment of an immune disorder, rheumatoid arthritis, lupus, systemic lupus erythematosus, or graft versus host disease.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOS: 32, 39, 40, 41, 42 and 43, respectively. In some embodiments, the antibody comprises VH of SEQ ID NO: 51 and VL of SEQ ID NO: 61. In some embodiments, the antibody comprises HC of SEQ ID NO: 73 and LC of SEQ ID NO: 83. Also provided is an antibody for use in therapy, for example in the treatment of an immune disorder, rheumatoid arthritis, lupus, systemic lupus erythematosus, or graft versus host disease.

- 17 043064

In some embodiments, the antibody is an agonist antibody.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, wherein the antibody competes for PD-1 binding with an antibody or antigen-binding fragment containing

VH with SEQ ID NO: 44 and VL with SEQ ID NO: 60;

VH c SEQ ID NO: 45 and VL c SEQ ID NO: 61;

VH c SEQ ID NO: 45 and VL c SEQ ID NO: 62;

VH c SEQ ID NO: 46 and VL c SEQ ID NO: 61;

VH c SEQ ID NO: 47 and VL c SEQ ID NO: 61;

VH c SEQ ID NO: 48 and VL c SEQ ID NO: 61;

VH c SEQ ID NO: 49 and VL c SEQ ID NO: 61;

VH c SEQ ID NO: 50 and VL c SEQ ID NO: 61; or

VH c SEQ ID NO: 51 and VL c SEQ ID NO: 61.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, wherein the antibody binds to the same epitope that binds to an antibody containing

VH with SEQ ID NO: 44 and VL with SEQ ID NO: 60;

VH c SEQ ID NO: 45 and VL c SEQ ID NO: 61;

VH c SEQ ID NO: 45 and VL c SEQ ID NO: 62;

VH c SEQ ID NO: 46 and VL c SEQ ID NO: 61;

VH c SEQ ID NO: 47 and VL c SEQ ID NO: 61;

VH c SEQ ID NO: 48 and VL c SEQ ID NO: 61;

VH c SEQ ID NO: 49 and VL c SEQ ID NO: 61;

VH c SEQ ID NO: 50 and VL c SEQ ID NO: 61; or

VH c SEQ ID NO: 51 and VL c SEQ ID NO: 61.

In some embodiments, the VH and VL regions or the HC and LC regions of an antibody that specifically binds to PD-1 or an antigen-binding fragment provided herein are encoded by a polynucleotide containing a polynucleotide sequence with

SEQ ID NOs: 52 and 63, respectively;

SEQ ID NO: 53 and 64, respectively;

SEQ ID NO: 53 and 65, respectively;

SEQ ID NO: 54 and 64, respectively;

SEQ ID NO: 55 and 64, respectively;

SEQ ID NOs: 56 and 64, respectively;

SEQ ID NO: 57 and 64, respectively;

SEQ ID NO: 58 and 64, respectively;

SEQ ID NO: 59 and 64, respectively;

SEQ ID NO: 74 and 85, respectively;

SEQ ID NO: 75 and 86, respectively;

SEQ ID NO: 75 and 87, respectively;

SEQ ID NO: 76 and 86, respectively;

SEQ ID NO: 77 and 86, respectively;

SEQ ID NO: 78 and 86, respectively;

SEQ ID NOs: 79 and 86, respectively;

SEQ ID NO: 80 and 86, respectively;

SEQ ID NO: 81 and 86, respectively;

SEQ ID NO: 136 and 137, respectively; or

SEQ ID NO: 138 and 139, respectively.

Antibodies of the PD1B512 line.

mAbs PD1B505, PD1B756, and PD1B757 are examples of antibodies from the mAb PD1B512 line. These mAbs have identical CDR regions with about 84% VH region identity and 90-99% VL region identity. mAbs of the PD1B512 line are non-ligand blocking. The line is characterized by HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 88, 89, 90, 91, 92 and 93, respectively, and the VH genomic sequence with SEQ ID NO: 122, and the VL genomic sequence with SEQ ID NO : 123.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and

- 18 043064

LCDR3 with SEQ ID NO: 88, 89, 90, 91, 92 and 93, respectively.

In some embodiments, the antibody or antigen-binding fragment thereof does not block the binding of PD-L1 to PD-1, and the absence of blocking is measured by the inability of the antibody to inhibit the clustering of PD-L1 expressing cells and PD-1 expressing cells as described in Example 1.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment provided herein, comprises a VH framework derived from IGHV2-5*04 (SEQ ID NO: 129).

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment provided herein, comprises a VL framework derived from IGKV2-28*01 (SEQ ID NO: 130).

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment provided herein, comprises a VH framework derived from IGHV2-5*04 (SEQ ID NO: 129) and a VL framework derived from IGKV2- 28*01 (SEQ ID NO: 130).

SEQ ID NO: 129 IGHV2-5*04

QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLALIYW

NDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTGTYYCV

SEQ ID NO: 130 IGKV2-28*01

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLG

SNRASGVPDRFSGSGSGTDFTLKISRVEEAEDVGVYYCMQALQTP

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising the heavy chain variable region (VH) of SEQ ID NO: 122. SEQ ID NO: 122 is the VH genomic sequence from mAb line PD1B512.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising the light chain variable region (VL) of SEQ ID NO: 123. SEQ ID NO: 123 is the VL genomic sequence from mAb line PD1B512.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising VH of SEQ ID NO: 122 and VL of SEQ ID NO: 123. CDRs are shown in bold in SEQ ID NO: 122 and SEQ ID NO: 123.

SEQ ID NO: 122 (genomic VH lineage PD1B112)

QXiTLKESGPX2LX3X4PX5QTLX6LTCX ₇ FSGFSLSTSGMGVSWIRQPX ₈ GKX ₉ LEW

LAHIYWDDDKRYXioPSLKSRLTIXnKDTSXi ₂ NQVXi3LXi ₄ Xi5TXi6Xi ₇ DXi ₈ Xi ₉ DTGTYY

CVRKGYYDYGYVMDYWGQGTX20VTVSS, and

X1 is V or I;

X ₂ is G or T;

X ₃ is L or V;

X ₄ is Q or K;

X ₅ represents S or T;

X6 is S or T;

X ₇ is S or T;

X8 is S or P;

X9 is G or A;

Hu is N or S;

Xc is S or T;

X ₁₂ is S or K;

X ₁₃ is F or V;

X ₁₄ is K or T;

X15 is I or M;

X ₁₆ is S or N;

X ₁₇ is V or M;

X ₁₈ is T or P;

X19 is A or V; And

X ₂₀ is T or L.

SEQ ID NO: 123 (genomic VL of line PD1B112)

DIVMTQX ₁ X ₂ LSX3PVTX ₄ GX ₅ X ₆ ASISCRSSKSLLHSNGITYLNWYLQKPGQSPQL

LIYQMSNLASGVPDRFSX ₇ SGSGTDFTLX ₈ ISRVEAEDVGVYYCAQNLELPTFGX ₉ GTK

X10EX11K, and

- 19 043064

X ₁ is A or S;

X2 is A or P;

X ₃ is N or L;

X ₄ is L or P;

X5 is T or E;

X6 is S or P;

X ₇ is S or G;

X ₈ represents R or K;

X ₉ is S or G;

X ₁₀ is L or V; And

X11 is M or I.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, contains a VH of SEQ ID NO: 94 or 95.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprises a VL of SEQ ID NO: 98, 99, or 100.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising a VH of SEQ ID NO: 94 or 95 and a VL of SEQ ID NO: 98, 99 or 100.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising VH of SEQ ID NO: 94 and VL of SEQ ID NO: 98. In some embodiments, the antibody comprises HC of SEQ ID NO: 104 and LC of SEQ ID NO: 108. An antibody is also provided for use in therapy, for example in the treatment of an immune disorder, rheumatoid arthritis, lupus, systemic lupus erythematosus, or graft versus host disease.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment provided herein, comprises VH of SEQ ID NO: 95 and VL of SEQ ID NO: 99. In some embodiments, the antibody comprises HC of SEQ ID NO: 105 and LC of SEQ ID NO: 109. An antibody is also provided for use in therapy, for example in the treatment of an immune disorder, rheumatoid arthritis, lupus, systemic lupus erythematosus, or graft versus host disease.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment provided herein, comprises VH of SEQ ID NO: 95 and VL of SEQ ID NO: 100. In some embodiments, the antibody comprises HC of SEQ ID NO: 105 and LC of SEQ ID NO: 110. An antibody is also provided for use in therapy, for example in the treatment of an immune disorder, rheumatoid arthritis, lupus, systemic lupus erythematosus, or graft versus host disease.

In some embodiments, the antibody is an agonist antibody.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, wherein the antibody competes for PD-1 binding with an antibody or antigen-binding fragment containing

VH of SEQ ID NO: 94 and VL of SEQ ID NO: 98;

VH c SEQ ID NO: 95 and VL c SEQ ID NO: 99; or

VH c SEQ ID NO: 95 and VL c SEQ ID NO: 100.

The invention also provides an isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, wherein the antibody binds to the same epitope that binds to an antibody containing

VH of SEQ ID NO: 94 and VL of SEQ ID NO: 98;

VH c SEQ ID NO: 95 and VL c SEQ ID NO: 99; or

VH c SEQ ID NO: 95 and VL c SEQ ID NO: 100.

In some embodiments, the VH and VL regions or the HC and LC regions of an antibody that specifically binds to PD-1 or an antigen-binding fragment provided herein are encoded by a polynucleotide containing a polynucleotide sequence with

SEQ ID NO: 96 and 101, respectively;

SEQ ID NOs: 97 and 102, respectively;

SEQ ID NO: 97 and 103, respectively;

SEQ ID NO: 106 and 111, respectively;

SEQ ID NOs: 107 and 112, respectively; or

SEQ ID NOs: 107 and 113, respectively.

Homologous antibodies and antibodies with conservative mutations.

Variants of antibodies that specifically bind to PD-1, or antigen-binding fragments provided herein, are within the scope of the invention. For example, options may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28 or 29

- 20 043064 amino acid substitutions in VH and/or VL, provided that the antibody variants retain or have improved properties compared to the original antibodies. In some embodiments, the sequence identity may be about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% c amino acid sequence VH and/or VL according to the invention. In some embodiments, the implementation of the variation is in the frame regions. In some embodiments, variants are generated by conservative substitutions.

For example, PD1B505 antibodies may contain substitutions at VH residue positions 1, 5, 6, 9, 16, 17, 20, 38, 43, 46, 57, 62, 63, 65, 69, 73, 76, 84, 85, 88, 93, 116 and/or 117 (residue numbering according to SEQ ID NO: 8) and at VL residue positions 1, 10, 11, 13, 15, 19, 21, 22, 29, 30, 43, 44, 46, 48, 59, 61, 71, 72, 73, 78, 79, 81, 84, 86, 101 and/or 107 (residue numbering according to SEQ ID NO: 14). Antibodies of the PD1B506 line may contain substitutions at VH residue positions 5, 7, 11, 12, 20, 38, 40, 48, 55, 56, 61, 62, 65, 66, 67, 68, 76, 82, 85, 87, 89, 91, 112 and/or 113 (residue numbering according to SEQ ID NO: 44) and at VL residue positions 3, 8, 9, 10, 11, 13, 16, 20, 21, 41, 42, 43, 46, 60, 63, 76, 77, 78, 80, 83, 85, 87, 100 and/or 106 (residue numbering according to SEQ ID NO: 60). PD1B512 lineage antibodies may contain substitutions at VH residue positions 2, 10, 12, 13, 15, 19, 23, 43, 46, 62, 72, 77, 81, 83, 84, 86, 87, 89, 90 and/or 117 and at VL residue positions 7, 8, 11, 15, 17, 18, 69, 79, 105, 109, and/or 111. Conservative substitutions can be made at any position indicated, and the resulting antibody variants tested for desired characteristics in assays, described in this document. In an alternative embodiment, substitutions in one mAb lineage can be made at the indicated positions by substituting the corresponding amino acid at a specific position present in other antibodies of that lineage.

Also provided are antibodies that specifically bind to PD-1, or antigen-binding fragments thereof containing VH and VL that are at least 80% identical:

VH with SEQ ID NO: 8 and VL with SEQ ID NO: 14;

VH c SEQ ID NO: 9 and VL c SEQ ID NO: 15;

VH c SEQ ID NO: 9 and VL c SEQ ID NO: 16;

VH c SEQ ID NO: 10 and VL c SEQ ID NO: 16;

VH c SEQ ID NO: 140 and VL c SEQ ID NO: 16;

VH c SEQ ID NO: 141 and VL c SEQ ID NO: 16;

VH c SEQ ID NO: 142 and VL c SEQ ID NO: 16;

VH c SEQ ID NO: 10 and VL c SEQ ID NO: 143;

VH c SEQ ID NO: 10 and VL c SEQ ID NO: 144;

VH c SEQ ID NO: 140 and VL c SEQ ID NO: 143;

VH c SEQ ID NO: 141 and VL c SEQ ID NO: 143;

VH c SEQ ID NO: 142 and VL c SEQ ID NO: 143;

VH c SEQ ID NO: 140 and VL c SEQ ID NO: 144;

VH c SEQ ID NO: 141 and VL c SEQ ID NO: 144;

VH c SEQ ID NO: 142 and VL c SEQ ID NO: 144;

VH c SEQ ID NO: 44 and VL c SEQ ID NO: 60;

VH c SEQ ID NO: 45 and VL c SEQ ID NO: 61;

VH c SEQ ID NO: 45 and VL c SEQ ID NO: 62;

VH c SEQ ID NO: 46 and VL c SEQ ID NO: 61;

VH c SEQ ID NO: 47 and VL c SEQ ID NO: 61;

VH c SEQ ID NO: 48 and VL c SEQ ID NO: 61;

VH c SEQ ID NO: 49 and VL c SEQ ID NO: 61;

VH c SEQ ID NO: 50 and VL c SEQ ID NO: 61;

VHcSEQIDNO: 51 and VL c SEQ ID NO: 61;

VH c SEQ ID NO: 94 and VL c SEQ ID NO: 98;

VH c SEQ ID NO: 95 and VL c SEQ ID NO: 99; or

VH c SEQ ID NO: 95 and VL c SEQ ID NO: 100.

In some embodiments, the identity is 85%. In some embodiments, the identity is 90%. In some embodiments, the identity is 91%. In some embodiments, the identity is 91%. In some embodiments, the identity is 92%. In some embodiments, the identity is 93%. In some embodiments, the identity is 94%. In some embodiments, the identity is 94%. In some embodiments, the identity is 96%. In some embodiments, the identity is 97%. In some embodiments, the identity is 98%. In some embodiments, the identity is 99%.

The percent identity between two sequences depends on the number of identical positions shared by the sequences (i.e., % identity=number of identical positions/total number of positions x100), taking into account the number of gaps and the length of each gap that must be built in to optimally align the two sequences.

The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput Appl. Biosci. 4:11-17 (1988)), which is built into the ALIGN program (version 2.0), using PAM120 residue mass tables, a gap length penalty of 12; and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)), which is built into the GAP program in GCG software package (available at http_//_www_gcg_com) using either Blossum 62 matrices or PAM250 matrices and gap weights of 16, 14, 12, 10, 8, 6 or 4 and gap length weights of 1, 2, 3, 4, 5 or 6.

In some embodiments, the antibody variants contain one or two conservative substitutions in any of the CDR regions, and the antibodies retain the desired properties of the original antibodies.

The term conservative modifications means amino acid modifications that do not significantly alter or affect the binding characteristics of an antibody containing such amino acid modifications. Conservative modifications include substitutions, additions and deletions of amino acids. Conservative amino acid substitutions are substitutions in which an amino acid is replaced by an amino acid residue having a similar side chain. Classes of amino acid residues having similar side chains are well defined and include amino acids with acidic side chains (eg, aspartic acid, glutamic acid), basic side chains (eg, lysine, arginine, histidine), non-polar side chains (eg, alanine, valine , leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g. glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g. phenylalanine, tryptophan, histidine, tyrosine) , aliphatic side chains (eg, glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (eg, asparagine, glutamine), beta-branched side chains (eg, threonine, valine, isoleucine), and sulfur-containing side chains ( cysteine, methionine). Additionally, any native residue in the polypeptide can be replaced with alanine, according to the method previously described as alanine-scanning mutagenesis (MacLennan et al. (1988) Acta Physiol. Scand. Suppl. 643:55-67; Sasaki et al. (1988) Adv. Biophys 35:1-24). Amino acid substitutions in the antibodies of the invention can be performed by known methods, for example by PCR-mediated mutagenesis (US patent No. 4683195). Alternatively, variant libraries can be generated, for example, by using random (NNK) or non-random codons, such as DVK codons encoding 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The characteristics of the resulting antibody variants can be examined using the assays described herein.

Engineered and modified antibodies.

Antibodies that specifically bind to PD-1, or antigen-binding fragments provided herein, can be further engineered to produce modified antibodies with similar or altered properties compared to the original antibodies. In the antibodies of the invention, VH, VL, VH, and VL regions, constant regions, a heavy chain framework, a light chain framework, or any or all of the six CDRs can be constructed.

Antibodies that specifically bind to PD-1, or antigen-binding fragments thereof, can be constructed by CDR grafting. One or more CDR sequences of antibodies of the invention can be grafted onto another framework sequence. CDR grafting can be performed using known methods and the methods described herein.

Suitable framework sequences can be obtained from public DNA databases or published sources, including germline antibody gene sequences. For example, the DNA sequences and encoded protein sequences of human germline light and heavy chain variable domain genes can be found in the international information system IMGT®, ImMunoGeneTics information system® http_//_www-imgt_org. Framework sequences that can be used to replace existing framework sequences in the antibodies of the invention may be those that show the highest percentage (%) identity with the original variable domains over the entire length of VH or VL or along the length of FR1, FR2, FR3 and FR4. In addition, acceptable scaffolds can be further selected based on the VH and VL in the CDR1 and CDR2 stretches or identical LCDR1, LCDR2, LCDR3, HCDR1 and HCDR2 of the canonical structure. Acceptable scaffolds can be selected using known methods such as human scaffold adaptation as described in US Pat. No. 8,748,356 or superhumanization as described in US Pat. No. 7,709,226.

- 22 043064

The framework sequences of the original and engineered antibodies can be further modified, for example, by backmutations, to restore and/or improve the binding of the resulting antibodies to the antigen, as described, for example, in US patent No. 6180370. The framework sequences of the original or engineered antibodies can be further modified by introducing mutations at one or more residues within the framework region (or alternatively within one or more CDR regions) to remove T-cell epitopes and therefore reduce the potential immunogenicity of the antibody. This approach is also referred to as deimmunization and is described in more detail in US Patent Publication No. US 20070014796.

Residues in the CDRs of the antibodies or antigen-binding fragments provided herein can be mutated to increase the affinity of the antibodies for PD-1.

Residues in the CDRs of antibodies or antigen-binding fragments provided herein can be mutated to minimize the risk of post-translational modifications. Amino acid residues of putative motifs for deamination (NS), acid-catalyzed hydrolysis (DP), isomerization (DS), or oxidation (W) can be substituted for any of the naturally occurring amino acids to mutagenesis the motifs, and the resulting antibodies can be tested for their functionality and stability using methods described in this document.

Antibodies that specifically bind to PD-1, or antigen-binding fragments provided herein, that are modified to improve stability, selectivity, cross-reactivity, affinity, immunogenicity or other desirable biological or biophysical properties are within the scope of the invention. Antibody stability is affected by a number of factors including:

(1) internal stacking of individual domains affecting their own stability;

(2) surface interactions of proteins affecting pairing of HC and LC;

(3) the depth of location of polar and charged residues;

(4) a network of H-bridges between polar and charged residues; and (5) surface charge and distribution of polar residues along with other intra- and intermolecular interactions (Worn et al. (2001) J. Mol. Biol. 305:989-1010).

Residues capable of destabilizing the structure can be identified based on the crystal structure of the antibody or, in certain cases, by molecular modeling, and the effect of residues on the stability of the antibody can be investigated by creating and evaluating variants that carry mutations in the identified residues. One way to increase the stability of an antibody is to increase the average transition temperature (Tm) as measured by differential scanning calorimetry (DSC). In general, the T _m of a protein is positively correlated with its stability and negatively correlated with its susceptibility to tertiary disruption and denaturation in solutions, as well as degradation, which depends on the propensity of the protein to disrupt tertiary structure (Remmele et al. (2000) Biopharm 13: 36-46). A number of studies have correlated the degree of physical stability of formulations, measured as thermal stability by DSC, with physical stability measurements made by other methods (Gupta et al. (2003) AAPS Pharm. Sci. 5E8; Zhang et al. (2004) J Pharm Sci 93:3076-89 Maa et al (1996) Int J Pharm 140:155-68 Bedu-Addo et al (2004) Pharm Res 21:1353-61 Remmele et al (1997) Pharm Res 15:200-8). The results of structural studies suggest that T _m Fab affects the long-term physical stability of the respective mAb.

Isotypes, allotypes of antibodies and Fc engineered antibodies.

Antibodies that specifically bind to PD-1, or antigen-binding fragments provided herein, can be of any known isotype or allotype, wild-type Fc, or engineered Fc.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment provided herein, is of the IgG1 isotype.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment provided herein, is of the IgG ₂ isotype.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment provided herein, is of the IgG ₃ isotype.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment provided herein, is of the IgG ₄ isotype.

Circulating endogenous carboxypeptidases can remove C-terminal lysine (CTL) from administered antibodies (Cai et al. (2011) Biotechnol. Bioeng. 108:404-412). Removal of CTL during production can be controlled below the maximum level by controlling the concentration of extracellular Zn ²⁺ , ethylenediaminetetraacetic acid (EDTA) or EDTA-Fe ³⁺ as described in US Patent Publication No. US 20140273092. CTL content in antibodies can be measured using known ways.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment provided herein, has a C-23043064 terminal lysine content of about 10% to about 90%. In some embodiments, the C-terminal lysine content is from about 20% to about 80%. In some embodiments, the C-terminal lysine content is from about 40% to about 70%. In some embodiments, the C-terminal lysine content is from about 55% to about 70%. In some embodiments, the C-terminal lysine content is about 60%.

The immunogenicity of therapeutic antibodies is associated with an increased risk of infusion reactions and a reduced duration of therapeutic response (Baert et al. (2003) N. Engl. J. Med. 348:602-08). The extent to which therapeutic antibodies induce an immune response in the host may be determined in part by the allotype of the antibody (Stickler et al. (2011) Genes and Immunity 12:213-21). The allotype of an antibody is associated with amino acid sequence variations at specific positions in the antibody constant region sequences. In table. 2 shows selected IgG1, IgG2 and IgG4 allotypes.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment provided herein, is of the GG2m(n) allotype.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment provided herein, is of the G2m(n-) allotype.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment provided herein, is of the G2m(n)/(n-) allotype.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment provided herein, is of the nG4m(a) allotype.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment provided herein, is of the G1m(17,1) allotype.

table 2

allotype Amino acid residue at the difference position (residue numbering: according to the EU catalog) IgG2 IgG4 IgGl 189 282 309 422 214 356 358 431 G2m(n) T M G2m(n-) R V G2m(n)/(n-) T V nG4m(a) L R glm(17) TO E M A Glm(17,l) TO D L A

To modulate antibody effector functions such as ADCC, antibody-dependent cellular phagocytosis (ADCP) and/or ADCP and/or pharmacokinetic properties, Fc mutations of antibodies that specifically bind to PD-1 or their antigen-binding fragments as provided herein can be administered. . This can be achieved by introducing mutation(s) into the Fc that modulates the binding of the mutant Fc to activating FcyR receptors (FcyRI, FcYRIIa, FcyRIII), inhibitory FcYRIIb and/or FcRn.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, contains at least one mutation in the Fc of the antibody.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, contains one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen mutations in fc.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, contains at least one Fc mutation that modulates the binding of the antibody to FcRn.

Fc positions that can be mutated to modulate the half-life of an antibody (e.g., FcRn binding) include positions 250, 252, 253, 254, 256, 257, 307, 376, 380, 428, 434, and 435. Examples mutations that can be introduced singly or in combination are T250Q, M252Y, I253A, S254T, T256E, P257I, T307A, D376V, E38OA, M428L, H433K, N434S, N434A, N434H, N434F, H435A, and H435R. Examples of mutations that can be introduced individually

- 24 043064 or in combination to increase the half-life of the antibody proposed herein are the mutations M428L/N434S, M252Y/S254T/T256E, T250Q/M428L, N434A and T307A/E380A/N434A.

Examples of mutations that can be introduced singly or in combination to decrease the half-life of an antibody provided herein are H435A, P257I/N434H,

D376V/N434H, M252Y/S254T/T256E/H433K/N434F, T308P/N434A and H435R.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, contains the M252Y/S254T/T256E mutation.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, contains at least one mutation in the Fc of the antibody that reduces the binding of the antibody to an Fcy activating receptor (FcyR) and/or reduces Fc effector functions, such as binding C1q, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or phagocytosis (ADCP).

Fc positions that can be mutated to reduce antibody binding to activating FcyR and consequently reduce effector function include positions 214, 233, 234, 235, 236, 237, 238, 265, 267, 268, 270, 295, 297 , 309, 327, 328, 329, 330, 331 and 365. Examples of mutations that can be introduced alone or in combination are mutations K214T, E233P, L234V, L234A, deletion G236, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, A330S and P331S in IgG1, IgG ₂ , IgG ₃ or IgG ₄ . Examples of mutation combinations that result in reduced ADCC antibody are L234A/L235A to IgG1, V234A/G237A/P238S/H268A/V309L/A330S/P331S to IgG ₂ , F234A/L235A to IgG ₄ , S228P/F234A /L235A in IgG ₄ , N297A in all Ig isotypes, V234A/G237A in IgG ₂ , K214T/E233P/L324V/L235A/G236-deletion/A327G/P331A/D365E/L358M in IgG ₁ , H268Q/V309L/A330S/ P331S to IgG ₂ , S267E/L328F to IgG1, L234F/L235E/D265A to IgG1, L234A/L235A/G237A/P238S/H268A/A330S/P331S to IgG1, S228P/F234A/L235A/G237A/P238S to IgG ₄ and S228P/F234A/L235A/ G236 deletion/G237A/P2388 in IgG ₄ . You can also use hybrid IgG ₂₄ Fc domains, such as Fc with residues 117-260 from IgG2 and residues 261-447 from IgG4.

An example of a mutation that results in reduced CDC antibodies is the K322A mutation.

The known S228P mutation can be performed to enhance IgG ₄ stability in IgG ₄ antibodies.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, contains a mutation at least one residue position 214, 233, 234, 235, 236, 237, 238, 265, 267, 268, 270, 295 , 297, 309, 322, 327, 328, 329, 330, 331 or 365.

In some embodiments, the antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, contains at least one mutation selected from the group consisting of K214T, E233P, L234V, L234A, deletions G236, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, K322, A330S and P331S.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, contains a mutation at least one residue position 228, 234, 235, 237, 238, 268, 322, 330, or 331.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, contains the K322A mutation.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, contains the S228P mutation.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, contains at least one mutation in the Fc of the antibody that enhances the binding of the antibody to the Fcy receptor (FcyR) and/or enhances Fc effector functions, such as C1q binding , complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and/or phagocytosis (ADCP).

Fc positions that can be mutated to increase antibody binding to activating FcyR and/or to enhance antibody effector functions include positions 236, 239, 243, 256, 290, 292, 298, 300, 305, 312, 326, 330, 332, 333, 334, 345, 360, 339, 378, 396 or 430 (numbering of residues corresponds to the EU catalog). Examples of mutations that can be introduced singly or in combination are: G236A mutation, S239D mutation, F243L mutation, T256A mutation, K290A mutation, R292P mutation, S298A mutation, Y300L mutation, V305L mutation, K326A mutation, A330K mutation, I3 mutation 32E, mutation E333A, mutation K334A, mutation A339T and mutation P396L. Examples of combinations of mutations that result in antibodies with increased ADCC or ADCP are: S239D/I332E mutation, S298A/E333A/K334A mutation, F243L/R292P/Y300L mutation, F243L/R292P/Y300L/P396L mutation, F243L/R29 mutation 2P/ Y300L/V305I/P396L and G236A/S239D/I332E mutation in IgG1.

Fc positions that can be mutated to enhance the CDC antibody include

- 25 043064 positions 267, 268, 324, 326, 333, 345 and 430. Examples of mutations that can be introduced individually or in combination are: S267E mutation, F1268F mutation, S324T mutation, K326A mutation, K326W mutation, E333A mutation, E345K mutation, E345Q mutation, E345R mutation, E345Y mutation, E430S mutation, E430F mutation and E430T mutation. Examples of combinations of mutations that lead to antibodies with increased CDC are: K326A/HIA mutation, K326W/E3333A mutation, H268F/S324T mutation, S267E/H268F mutation, S267E/S324T mutation and mutation S267E/H268F/S324T in IgG1.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, contains at least one mutation in the Fc region that enhances the binding of the antibody to FcyRIIb. Enhanced binding to FcyRIIb can lead to downregulation of FcyRIIb B cells and/or lead to antibody clustering and subsequent activation of downstream PD-1 signaling pathways.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, contains the S267E mutation.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, contains the S267D mutation.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, contains the S267E/I332E mutation.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, contains the S267E/L328F mutation.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, contains the G236D/S267E mutation.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, contains the mutation P238D/E233D/G237D/H268D/P271G/A330R.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, contains the P238D mutation.

Antibody-dependent cellular cytotoxicity, antibody-dependent cellular cytotoxicity, or ADCC, is a cell death induction mechanism that depends on the interaction of antibody-coated target cells with effector cells with lytic activity, such as natural killer (NK) cells, monocytes, macrophages, and neutrophils, through Fc gamma receptors (FcyR) expressed on effector cells. For example, NK cells express FcyRIIIa while monocytes express FcyRI, FcyRII and FcyRIIIa. The ADCC activity of the antibodies provided herein can be assessed by an in vitro assay using PD-1 expressing cells as target cells and NK cells as effector cells. Cytolysis can be detected by the release of a label (eg, radioactive substrates, fluorescent dyes, or naturally occurring intracellular proteins) from lysed cells. In the assay example, target cells were used at a ratio of 1 target cell to 4 effector cells. Target cells are prelabeled with BATDA and combined with effector cells and antibody of interest. Samples are incubated for 2 hours and cell lysis is measured by measuring the release of BATDA into the supernatant. Data are normalized for maximum cytotoxicity with 0.67% Triton X-100 (Sigma Aldrich) and minimum control as determined by BATDA release from target cells in the absence of any antibody.

Antibody-dependent cellular phagocytosis (ADCP) refers to a mechanism for killing antibody-coated target cells by internalization by phagocytic cells such as macrophages or dendritic cells. ADCP can be assessed using monocytic macrophages as effector cells and Dowdy cells (ATCC® CCL-213™) or any other PD-1 expressing cells as target cells engineered to express green fluorescent protein (GFP) or other labeling molecule . In an example assay, the ratio of effector cells to target cells can be, for example, 4:1. Effector cells can be incubated with target cells for 4 hours with or without an antibody of the invention. After incubation, cells can be separated using accutase. Identification of macrophages can be done with anti-CD11b and anti-CD14 antibodies associated with a fluorescent label, and the percentage of phagocytosis can be determined based on the % fluorescent GFP in CD11+CD14+ macrophages using standard methods.

Complement-dependent cytotoxicity, or CDC, refers to a cell death induction mechanism in which the Fc effector domain of a target-bound antibody binds and activates the C1q complement component, which in turn activates the complement cascade leading to death of the target cell. Complement activation can also lead to the deposition of complement components on the surface of target cells, which facilitates CDC by binding complement receptors (eg, CR3) on leukocytes. CDC for cells can be measured, for example, by seeding Daudi cells at 1x105 cells/well (50 µl/well) in RPMI-B (RPMI supplemented with 1% BSA), adding 50 µl of test antibodies to the wells to a final concentration in the range of 0- 100

- 26 043064 µg/ml, incubating the reaction mixture for 15 min at room temperature, adding 11 µl of pooled human serum to the wells and incubating the reaction mixture for 45 min at 37°C. The percentage (%) of lysed cells can be determined as the % of cells stained with propidium iodide in a FACS analysis using standard methods.

Antibody binding to FcyR or FcRn can be assessed using flow cytometry on cells engineered to express each of the receptors. In the binding assay example, 2x105 cells per well were plated in a 96-well plate and blocked with bovine serum albumin (BSA) staining buffer (BD Biosciences, San Jose, USA) for 30 minutes at 4°C. Cells are incubated with test antibody on ice for 1.5 h at 4°C. After washing twice with BSA staining buffer, the cells were incubated with R-phycoerythrin (R-PE) labeled anti-human IgG secondary antibody (Jackson Immunoresearch Laboratories) for 45 min at 4°C. Cells were washed twice in staining buffer and then resuspended in 150 µl of staining buffer containing a 1:200 dilution of live/dead DRAQ7 dye (Cell Signaling Technology, Danvers, USA). PE and DRAQ7 signals from stained cells were determined using a Miltenyi_MACSQuant flow cytometer (Miltenyi Biotec, Auburn, USA) using channels B2 and B4, respectively. Live cells are gated with DRAQ7 exclusion and geometric mean fluorescence signals are determined for at least 10,000 collected live events. For analysis, FlowJo software (Tree Star) was used. The data are plotted as the logarithm of antibody concentration versus mean fluorescence signals. Conduct a nonlinear regression analysis.

The term enhance or enhanced refers to enhanced effector function (e.g. ADCC, CDC and/or ADCP) or increased binding to the Fcγ receptor (FcyR) or FcRn receptor of an antibody of the invention having at least one mutation in the Fc region compared to original antibody without mutation. The term enhanced may mean an increase of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more, or a statistically significant increase.

The term decreased or reduced refers to decreased effector function (e.g. ADCC, CDC and/or ADCP) or reduced binding to the Fcy receptor (FcyR) or FcRn receptor of an antibody of the invention having at least one mutation in the Fc region compared to original antibody without mutation. The term decrease may mean a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more, or a statistically significant decrease.

The term modulate refers to increased or decreased effector function (e.g. ADCC, CDC and/or ADCP) or increased or decreased binding to the Fcy receptor (FcyR) or FcRn receptor of an antibody of the invention having at least one mutation in the Fc region of compared to the original antibody without the mutation.

glycomodified antibodies.

The ability of monoclonal antibodies to induce ADCC can be enhanced by designing their oligosaccharide component. Human IgG1 or IgG ₃ undergo N-glycosylation at Asn297 by most glycans in the well-known 2-antennary forms G0, G0F, G1, G1F, G2 or G2F. Antibodies produced by N-engineered CHO cells typically have a glycan fucose content of at least about 85%. Removal of the central fucose from type 2-anthenary complex oligosaccharides attached to Fc regions enhances antibody ADCC through improved FcyRIIIa binding without altering antigen binding or CDC activity. Such mAbs can be generated using a variety of methods that have been reported to result in successful expression of relatively highly defucosylated antibodies bearing Fc-oligosaccharides of type 2-anthenary complex, such as controlling the osmolality of the culture medium (Konno et al., Cytotechnology 64 :249-65, 2012), use as host cell line of variant CHO line Lecl3 (Shields et al., J. Biol. Chem. 277:26733-26740, 2002), use as host cell line of variant cell line CHO EB66 (Olivier et al., MAbs; 2(4):405-415, 2010; PMID:20562582), using the YB2/0 rat hybridoma cell line as a host cell line (Shinkawa et al., J. Biol. Chem. 278:3466-3473, 2003), introduction of small interfering RNA specific for the 1,6-fucosyltransferase (FUT8) gene (Mori et al., Biotechnol Bioeng 88:901-908, 2004), or co-expression of e-1,4- Nacetylglucosaminyltransferase III and α-mannosidase II of the Golgi complex, or use of the strong inhibitor of α-mannosidase I, kyfunesin (Ferrara et al., J. Biol. Chem. 281:5032-5036, 2006, Ferrara et al., Biotechnol. Bioeng. 93:851-861, 2006; Xhou et al., Biotechnol. Bioeng. 99:652-65, 2008).

In some embodiments described herein, antibodies of the invention have a 2-antennary glycan structure with a fucose content of about 1% to about 15%, such as about 15, 14, 13, 12, 11, 10, 9, 8, 7 , 6, 5, 4, 3, 2 or 1%. In some embodiments, the antibodies of the invention have a glycan structure with a fucose content of about 50%, 40%, 45%, 40%, 35%, 30%, 25%, or 20%.

The term fucose content means the amount of fucose monosaccharide within the saccharide

- 27 043064 chains in position Asn297. The relative amount of fucose is the percentage of fucose-containing structures related to all glycostructures. They can be characterized and quantified in a variety of ways, for example:

1) using time-of-flight matrix-assisted laser desorption/ionization (MALDI-TOF) of a sample treated with N-glycosidase F (eg complex, hybrid, oligomannose and high-mannose structures) as described in International Patent Application No. WO 2008/0775462);

2) via enzymatic release of Asn297 glycans followed by derivatization and detection/quantitation by high performance liquid chromatography (HPLC) (Ultra-Performance Liquid Chromatography (UHPLC)) with fluorescence detection and/or High-Performance Liquid Chromatography with Mass Spectrometry (HPLC-MS) (Ultra-Performance liquid chromatography with mass spectrometry (UHPLC-MS));

3) analysis of intact protein of native or reconstituted mAb with or without treatment of Asn297 glycans with Endo S enzyme or other enzyme that cleaves the bond between the first and second GlcNAc monosaccharides, keeping the fucose attached to the first GlcNAc;

4) cleavage of the mAb into its constituent peptides by enzymatic cleavage (eg trypsin or Lys-C endopeptidase) and subsequent separation, detection and quantification by HPLC-MS (UHPLC-MS);

5) separating the mAb oligosaccharides from the mAb protein by specific enzymatic deglycosylation from PNGase F to Asn 297.

The oligosaccharides released in this way can be fluorophore-labeled, separated and identified by various auxiliary methods that allow: to accurately characterize glycan structures by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry by comparing experimental masses with theoretical masses, to determine the degree of sialylation using ion-exchange HPLC (GlycoSep C), separate and quantify oligosaccharide forms by hydrophilicity using ordinary phase HPLC (GlycoSep N), and separate and quantify oligosaccharide forms using high performance laser-induced fluorescence capillary electrophoresis (HPCE-LIF).

As used herein, the expressions low fucose or low fucose refer to antibodies with a fucose content of about 1-15%.

Normal fucose or normal fucose refers herein to antibodies with a fucose content greater than about 50%, typically greater than about 80%, or greater than about 85%.

Anti-idiotypic antibodies.

Anti-idiotypic antibodies are antibodies that specifically bind to the antibodies described herein that specifically bind to PD-1.

The invention also provides an anti-idiotypic antibody that specifically binds to antibodies provided herein that specifically bind to PD-1.

The invention also provides an anti-idiotypic antibody that specifically binds to an antibody containing:

- 28 043064

VH c SEQ ID NO: 8 and VL c SEQ ID NO: 14;

VH c SEQ ID NO: 9 and VL c SEQ ID NO: 15;

VH c SEQ ID NO: 9 and VL c SEQ ID NO: 16;

VH c SEQ ID NO: 10 and VL c SEQ ID NO: 16;

VH c SEQ ID NO: 140 and VL c SEQ ID NO: 16;

VH c SEQ ID NO: 141 and VL c SEQ ID NO: 16;

VH c SEQ ID NO: 142 and VL c SEQ ID NO: 16;

VH c SEQ ID NO: 10 and VL c SEQ ID NO: 143;

VH c SEQ ID NO: 10 and VL c SEQ ID NO: 144;

VH c SEQ ID NO: 140 and VL c SEQ ID NO: 143;

VH c SEQ ID NO: 141 and VL c SEQ ID NO: 143;

VH c SEQ ID NO: 142 and VL c SEQ ID NO: 143;

VH c SEQ ID NO: 140 and VL c SEQ ID NO: 144;

VH c SEQ ID NO: 141 and VL c SEQ ID NO: 144;

VH c SEQ ID NO: 142 and VL c SEQ ID NO: 144;

VH c SEQ ID NO: 44 and VL c SEQ ID NO: 60;

VH c SEQ ID NO: 45 and VL c SEQ ID NO: 61;

VH c SEQ ID NO: 45 and VL c SEQ ID NO: 62;

VH c SEQ ID NO: 46 and VL c SEQ ID NO: 61;

VH c SEQ ID NO: 47 and VL c SEQ ID NO: 61;

VH c SEQ ID NO: 48 and VL c SEQ ID NO: 61;

VH c SEQ ID NO: 49 and VL c SEQ ID NO: 61;

VH c SEQ ID NO: 50 and VL c SEQ ID NO: 61;

VH c SEQ ID NO: 51 and VL c SEQ ID NO: 61;

VH c SEQ ID NO: 94 and VL c SEQ ID NO: 98;

VH c SEQ ID NO: 95 and VL c SEQ ID NO: 99; or

VH c SEQ ID NO: 95 and VL c SEQ ID NO: 100.

An anti-idiotypic (Id) antibody is an antibody that recognizes the antigenic determinants (eg, paratope or CDR) of an antibody. The Id antibody may be blocking or non-blocking antigen. An antigen-blocking Id antibody can be used to detect a free antibody in a sample (eg, the anti-PD-1 antibody of the invention described herein). A non-blocking Id antibody can be used to detect all antibodies (free, partially bound to an antigen, or fully bound to an antigen) in a sample. An Id antibody can be obtained by immunizing an animal with an antibody for which an anti-Id antibody is obtained.

An anti-Id antibody can also be used as an immunogen to induce an immune response in another animal, producing a so-called anti-anti-Id antibody. The anti-anti-Id antibody may be epitopically identical to the primary mAb that induced the anti-Id. Thus, by using antibodies to the idiotypic determinants of the mAb, other clones expressing antibodies of identical specificity can be identified. Anti-idiotypic antibodies can be variated (thereby anti-Id variants) and/or derivatized using any suitable technique, such as those described elsewhere herein for antibodies that specifically bind PD-1.

Conjugates of antibodies provided herein that specifically bind PD-1.

The invention also provides an immunoconjugate containing an isolated antibody or antigen-binding fragment thereof that specifically binds to PD-1 conjugated to a heterologous molecule.

In some embodiments, the heterologous molecule is a detectable label or cytotoxic agent.

The invention also provides an antibody, or an antigen-binding fragment thereof, that specifically binds to PD-1, conjugated to a detectable label.

The invention also provides an antibody, or an antigen-binding fragment thereof, that specifically binds to PD-1, conjugated to a cytotoxic agent.

Antibodies or antigen-binding fragments thereof that bind to PD-1 can be used to target therapeutic agents to cells expressing PD-1. Cells, such as activated T cells that overexpress PD-1, can be targeted by an antibody that specifically binds PD-1 conjugated to a cytotoxic agent that kills the cell upon internalization of the anti-PD-1 antibody. Alternatively, cells expressing PD-1 may be targets for an anti-PD-1 antibody linked to a therapeutic agent designed to modify cellular function after internalization.

In some embodiments, the detectable label is also a cytotoxic agent.

An isolated antibody provided herein that specifically binds to PD-1, or an antigen-binding fragment thereof, conjugated to a detectable label, can be used to evaluate PD-1 expression in various assays.

Detectable labels include compositions that, when conjugated to an isolated antibody provided herein that specifically binds to PD-1, or an antigen-binding fragment thereof, renders it detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.

Examples of detectable labels include radioactive isotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron dense reagents, enzymes (e.g. widely used in enzyme immunoassay (ELISA)), biotin, digoxigenin, haptens, luminescent molecules, chemiluminescent molecules, fluorochromes, fluorophores, fluorescence quenching agents, colored molecules, radioactive isotopes, scintillators, avidin, streptavidin, protein A, protein G, antibodies or their fragments, polyhistidine, Ni ²⁺ , Flag markers, myc markers, heavy metals, enzymes, alkaline phosphatase, peroxidase, luciferase, electron donors/acceptors, acridinium esters and colorimetric substrates.

The detectable label may emit a signal spontaneously, for example, when the detectable label is a radioactive isotope. In other cases, the detectable label emits a signal as a result of stimulation by an external field.

Examples of radioactive isotopes can be y-emitting, Auger electron-emitting, β-emitting, α-emitting or positron-emitting radioactive isotopes. Examples of radioactive isotopes include ³ H, ¹¹ C, ¹³ C, ¹⁵ N, ¹⁸ F, ^{19 F, 55} Co, ⁵⁷ Co, ⁶⁰ Co, ⁶¹ Cu, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁸ Ga, ^{72 As} , ⁷⁵ Br, ⁸⁶ Y, ⁸⁹ Zr, ⁹⁰ Sr, ^94m Tc, ^99m Tc, ¹¹⁵ In, ¹²³ I, ¹²⁴ 1, ¹²⁵ I, ¹³¹ 1, ²¹¹ At, ²¹² Bi, ²¹³ Bi, ²²³ Ra, ²²⁶ Ra, ²²⁵ Ac and ²²⁷ As.

Examples of metal atoms include metals with an atomic number greater than 20, such as calcium atoms, scandium atoms, titanium atoms, vanadium atoms, chromium atoms, manganese atoms, iron atoms, cobalt atoms, nickel atoms, copper atoms, zinc atoms, gallium atoms, atoms germanium, arsenic atoms, selenium atoms, bromine atoms, krypton atoms, rubidium atoms, strontium atoms, yttrium atoms, zirconium atoms, niobium atoms, molybdenum atoms, technetium atoms, ruthenium atoms, rhodium atoms, palladium atoms, silver atoms, cadmium atoms, indium atoms, tin atoms, antimony atoms, tellurium atoms, iodine atoms, xenon atoms, cesium atoms, barium atoms, lanthanum atoms, hafnium atoms, tantalum atoms, tungsten atoms, rhenium atoms, osmium atoms, iridium atoms, platinum atoms, gold atoms , mercury atoms, thallium atoms, lead atoms, bismuth atoms, francium atoms, radium atoms, actinium atoms, cerium atoms, praseodymium atoms, neodymium atoms, promethium atoms, samarium atoms, europium atoms, gadolinium atoms, terbium atoms, dysprosium atoms, atoms holmium, erbium atoms, thulium atoms, ytterbium atoms, lutetium atoms, thorium atoms, protactinium atoms, uranium atoms, neptunium atoms, plutonium atoms, americium atoms, curium atoms, berkelium atoms, californium atoms, einsteinium atoms, fermium atoms, mendelevium atoms, nobelium atoms or lawrencium atoms.

In some embodiments, the metal atoms may be alkaline earth metals with an atomic number greater than twenty.

In some embodiments, the metal atoms may be lanthanides.

In some embodiments, the metal atoms may be actinides.

In some embodiments, the metal atoms may be transition metals.

In some embodiments, the metal atoms may be post-transition metals.

In some embodiments, the metal atoms may be gold atoms, bismuth atoms, tantalum atoms, and gadolinium atoms.

In some embodiments, the metal atoms may be metals with atomic number 53 (i.e., iodine) to 83 (i.e., bismuth).

In some embodiments, the implementation of the metal atoms may be atoms that are acceptable for magnetic resonance imaging.

Metal atoms can be metal ions with an oxidation state of +1, +2, +3, for example Ba ²⁺ , Bi ³⁺ , Cs ⁺ , Ca ²⁺ , Cr ²⁺ , Cr ³⁺ , Cr ⁶⁺ , Co ²⁺ , Co ³⁺ , Cu ⁺ , Cu ²⁺ , Cu ³⁺ , Ga ³⁺ , Gd ³⁺ , Au ⁺ , Au ³⁺ , Fe ²⁺ , Fe ³⁺ , F ³⁺ , Pb ²⁺ , Mn ²⁺ , Mn ³⁺ , Mn ⁴⁺ , Mn ⁷⁺ , Hg ²⁺ , Ni ²⁺ , Ni ³⁺ , Ag ⁺ , Sr ²⁺ , Sn ²⁺ , Sn ⁴⁺ and Zn ²⁺ . The metal atoms may include a metal oxide such as iron oxide, manganese oxide, or gadolinium oxide.

Suitable dyes include any commercially available dyes such as, for example, 5(6)-carboxyfluorescein, maleimide IRDye 680RD or IRDye 800CW, dyes based on

- 30 043064 ruthenium/polypyridyl complexes, etc.

Acceptable fluorophores include fluorescein isothiocyanate (FITC), fluorescein thiosemicarbazide, rhodamine, Texas Red, CyDye dyes (e.g., Cy3, Cy5, Cy5.5), Alexa Fluor dyes (e.g., Alexa488, Alexa555, Alexa594; Alexa647), near fluorescent dyes IR range (NIR) (700-900 nm) and carbocyanine and aminostyrene dyes.

The isolated antibody provided herein that specifically binds to PD-1, or an antigen-binding fragment thereof, conjugated to a detectable label, can be used as an imaging agent.

In some embodiments, an isolated antibody as provided herein that specifically binds to PD-1, or an antigen-binding fragment thereof, is conjugated to a cytotoxic agent.

In some embodiments, the cytotoxic agent is a chemotherapeutic drug, a drug, a growth inhibitory agent, a toxin (e.g., a bacterial, fungal, plant, or animal toxin with enzymatic activity, or fragments thereof), or a radioactive isotope (i.e., a radioactive conjugate ).

In some embodiments, the cytotoxic agent is daunomycin, doxorubicin, methotrexate, vindesine, bacterial toxins such as diphtheria toxin, ricin, geldanamycin, maytansinoids, or calicheamicin. A cytotoxic agent can induce cytotoxic and cytostatic effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

In some embodiments, the cytotoxic agent is a toxin with enzymatic activity, such as diphtheria toxin A chain, diphtheria toxin non-binding active fragments, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modecin, alpha-sarcin, Aleurites fordii proteins, diantine proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinali inhibitor, gelonin, mitogellin, restrictocin, fenomycin, enomycin and tricothecenes.

In some embodiments, the implementation of the cytotoxic agent is a radionuclide, such as ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y and ¹⁸⁶ Re.

In some embodiments, the cytotoxic agent is dostatins or peptide analogs and derivatives of dolastatin, auristatin, or monomethylauristatin phenylalanine. Example molecules are described in US Pat. ) and have anticancer and antifungal activity. The drug functional group of dostatin or auristatin can be attached to an antibody of the invention, via the N (amino) terminus or C (carboxyl) terminus of the peptide drug functional group (WO 02/088172), or via any cysteine artificially introduced into the antibody.

An isolated antibody provided herein that specifically binds to PD-1, or an antigen-binding fragment thereof, can be conjugated to a detectable label using known methods.

In some embodiments, the detectable label is complexed with a chelating agent.

In some embodiments, a detectable label is conjugated to an isolated antibody as provided herein that specifically binds to PD-1, or an antigen-binding fragment thereof, via a linker.

A detectable label or cytotoxic fragment can be directly or indirectly linked to an antibody provided herein that specifically binds to PD-1, or an antigen-binding fragment thereof, using known methods. Suitable linkers known in the art include, for example, prosthetic groups, non-phenolic linkers (derivatives of N-succinimidyl benzoates; dodecarborate), chelating groups of both macrolytic and acyclic chelators, for example 1,4,7,10-tetraazacyclododecan-1 derivatives ,4,7,10,tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA) derivatives, S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid derivatives (NOTA) and 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA) derivatives, N-succinimidyl-3-(2pyridyldithiol) propionate (SPDP), iminothiolane (IT), dysfunctional imidoester derivatives ( such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis-(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-( p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorides (such as 1,5difluoro-2,4-dinitrobenzene) and other chelating groups. Suitable peptide linkers are well known.

In some embodiments, an antibody provided herein that

- 31 043064 binds specifically to PD-1, or its antigen-binding fragment is removed from the blood by the kidneys.

Sets.

The invention also provides a kit containing an antibody described herein that specifically binds to PD-1, or an antigen-binding fragment thereof.

The kit can be used for therapeutic applications and as diagnostic kits.

The kit can be used to detect the presence of PD-1 in a sample.

In some embodiments, the kit comprises an antibody as provided herein that specifically binds to PD-1, or an antigen-binding fragment thereof, and reagents for detecting the antibody. The kit may contain one or more of the other elements: instructions for use; other reagents, such as a label, a therapeutic agent or an agent used for chelation or other combination, an antibody for labeling, or a therapeutic agent, or a radioprotective composition; devices or other materials for preparing the antibody for administration; pharmaceutically acceptable carriers and devices or other materials for administration to a patient.

In some embodiments, a kit contains an antibody as provided herein that specifically binds to PD-1, or an antigen-binding fragment thereof, in a container, and instructions for using the kit.

In some embodiments, the antibody in the kit is labeled.

In some embodiments, the kit contains an antibody that specifically binds PD1, or an antigen-binding fragment thereof, containing

V H with SEQ ID NO: 8 and VL with SEQ ID NO: 14;

V H c SEQ ID NO: 9 and VL c SEQ ID NO: 15;

V H c SEQ ID NO: 9 and VL c SEQ ID NO: 16;

VH c SEQ ID NO: 10 and VL c SEQ ID NO: 16;

VH c SEQ ID NO: 140 and VL c SEQ ID NO: 16;

VH c SEQ ID NO: 141 and VL c SEQ ID NO: 16;

VH c SEQ ID NO: 142 and VL c SEQ ID NO: 16;

VH c SEQ ID NO: 10 and VL c SEQ ID NO: 143;

VH c SEQ ID NO: 10 and VL c SEQ ID NO: 144;

VH c SEQ ID NO: 140 and VL c SEQ ID NO: 143;

VH c SEQ ID NO: 141 and VL c SEQ ID NO: 143;

VH c SEQ ID NO: 142 and VL c SEQ ID NO: 143;

VH c SEQ ID NO: 140 and VL c SEQ ID NO: 144;

VH c SEQ ID NO: 141 and VL c SEQ ID NO: 144;

VH c SEQ ID NO: 142 and VL c SEQ ID NO: 144;

VH with SEQ ID NO: 44 and VL with SEQ ID NO 60; VH c SEQ ID NO: 45 and VL c SEQ ID NO 61; VH c SEQ ID NO: 45 and VL c SEQ ID NO 62; VH c SEQ ID NO: 46 and VL c SEQ ID NO 61; VH c SEQ ID NO: 47 and VL c SEQ ID NO 61; VH c SEQ ID NO: 48 and VL c SEQ ID NO 61; VH c SEQ ID NO: 49 and VL c SEQ ID NO 61; VH c SEQ ID NO: 50 and VL c SEQ ID NO 61; VH c SEQ ID NO: 51 and VL c SEQ ID NO 61; VH c SEQ ID NO: 94 and VL c SEQ ID NO 98; VH c SEQ ID NO: 95 and VL c SEQ ID NO 99; or VH c SEQ ID NO: 95 and VL c SEQ ID NO 100.

Ways to detect PD-1.

The invention also provides a method for detecting PD-1 in a sample, which includes obtaining a sample, bringing the sample into contact with an antibody described herein that specifically binds PD-1, or an antigen-binding fragment thereof, and detecting an antibody associated with PD-1 in the sample. .

In some embodiments, the sample may be obtained from urine, blood, blood serum, blood plasma, saliva, ascitic fluid, circulating cells, synovial fluid, circulating cells, cells not associated with tissues (i.e. free cells), tissues (e.g. , surgically resected tissue, biopsy materials, including those obtained using fine needle aspiration biopsy), histological preparations, and the like.

- 32 043064

Antibodies or antigen-binding fragments of the invention associated with PD-1 can be detected using known methods. Examples of methods include direct labeling of antibodies using fluorescent or chemiluminescent labels or radioactive labels or attaching an easily detectable functional group such as biotin, enzymes or epitope labels to the antibodies of the invention. Examples of labels and functional groups are ruthenium, ¹¹¹ In-DOTA, ¹¹¹ In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, polyhistidine (HIS label), acridine dyes, cyanine dyes, fluorone dyes, oxazine dyes , phenanthridine dyes, rhodamine dyes and Alexafluor® dyes.

The antibodies of the invention can be used in a number of assays to detect PD-1 in a sample. Examples of assays are Western blot analysis, radioimmunoassay, surface plasmon resonance, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescent (ECL) immunoassay, immunohistochemistry, fluorescence activated cell sorting (FACS), or solid phase ELISA.

Methods for obtaining antibodies.

Antibodies provided herein that specifically bind to PD-1, or antigen-binding fragments thereof, can be produced using a variety of technologies. For example, the hybridoma method of Kohler and Milstein can be used to generate monoclonal antibodies. In the hybridoma method, a mouse or other animal host, such as a hamster, rat, or chicken, is immunized with human and/or cynomolgus monkey PD-1 antigens, such as the extracellular domain of PD-1, followed by fusion of splenocytes from immunized animals with myeloma cells using standard methods with the formation of hybridoma cells. Colonies arising from single cells of an immortalized hybridoma can be screened for the production of antibodies with desirable properties such as binding specificity, cross-reactivity, antigen affinity, and functionality, such as agonist activity.

Examples of humanization techniques involving the selection of human acceptor scaffolds include CDR grafting (US Pat. No. 5,225,539), SDR grafting (US Pat. No. 6,818,749), resurfacing (Padlan, (1991) Mol. Immunol. 28:489-499 ), resurfacing of specificity-determining residues (US Patent Publication No. 2010/0261620), human scaffold adaptation (US Patent No. 8748356), or superhumanization (US Patent No. 7709226). In these methods, the CDRs or a portion of the CDR residues of the parent antibodies are transferred to human scaffolds, which can be selected based on their overall homology to the parent scaffolds, CDR length similarity, or canonical structure identity, or a combination of both.

Humanized antibodies can be further optimized to improve their selectivity or affinity for the desired antigen by incorporating altered scaffold-supporting residues while retaining binding affinity (backmutations) by techniques such as those described in International Patent Publication Nos. WO 1090/007861 and WO 1992/22653 , or by inserting a variation into any of the CDRs, for example, to improve the affinity of the antibody.

To obtain antibodies to PD-1, transgenic animals carrying human immunoglobulin (Ig) loci in their genome, such as mice, rats or chickens, can be used, which are described, for example, in US patent No. 6150584, international patent publication No. WO 1999/45962 , International Patent Publication No. WO 2002/066630, WO 2002/43478, WO 2002/043478 and WO 1990/04036. Endogenous immunoglobulin loci in such animals can be ruptured or removed, and at least one complete or partial human immunoglobulin locus can be inserted into the animal's genome by homologous or non-homologous recombination, using transchromosomes, or using minigenes. To obtain human antibodies directed against a selected antigen using the technology described above, companies such as Regeneron (http://_www_regeneron_com), Harbor Antibodies (http://_www_harbourantibodies_com), Open Monoclonal Technology, Inc. can be consulted. (OMT) (http://_www_omtinc_net), KyMab (http://_www_kymab_com), Trianni (http://_www.trianni_com) and Ablexis (http://_www_ablexis_com).

Antibodies can be selected from a phage display library, wherein the phage is designed to express human immunoglobulins or portions thereof, such as Fabs, single chain antibodies (scFv), or unpaired or paired antibody variable regions. Antibodies of the invention can be isolated, for example, from a phage display library expressing antibody heavy and light chain variable regions as fusion proteins with the bacteriophage coat protein pIX, as described by Shi et al. (2010) J. Mol. Biol. 397:385-96 and International Patent Publication No. WO09/085462). Libraries can be screened for phage binding to human and/or cynomolgus monkey PD-1, and the resulting positive clones can be further characterized, Fab can be isolated from clone lysates and expressed as full-length IgG.

The production of immunogenic antigens and the production of monoclonal antibodies can be performed using any suitable technique, such as production of a recombinant protein. Immunogenic antigens can be administered to animals in the form of purified protein or protein mixtures, including whole cells or cell or tissue extracts, or the antigen can be formed de novo in the animal body from nucleic acids encoding the specified antigen or part thereof.

In some embodiments, an antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, of the invention is a bispecific antibody.

In some embodiments, the antibody or antigen-binding fragment thereof of the invention is a multispecific antibody.

Proposed in the invention monospecific antibodies that specifically bind to PD-1 can be designed as bispecific antibodies, which are also included in the scope of the present invention.

Full-length bispecific antibodies can be generated, for example, by exchanging Fab arms (e.g., by exchanging half molecules, exchanging one heavy chain-light chain pair) between two monospecific divalent antibodies, introducing substitutions into the CH3 interface of the heavy chain in each hemimolecule that promote the formation of a heterodimer from two half-molecules of antibodies having different specificities, either in vitro in a cell-free environment, or using co-expression. The Fab-arm exchange reaction is the result of disulfide isomerization and dissociation-association of CH3 domains. Heavy chain disulfide bonds are restored in the hinge regions of the original monospecific antibodies. The obtained free cysteines of one of the original monospecific antibodies form a disulfide bond of heavy chains with cysteine residues of the second molecule of the original monospecific antibody, and simultaneously the CH3 domains of the original antibodies are released and rearranged by dissociation-association. Fab arm CH3 domains can be designed to allow for heterodimerization rather than homodimerization. The resulting product is a bispecific antibody having two Fab arms or hemimolecules, each of which binds to a separate epitope.

Bispecific antibodies can also be generated using regimens such as Triomab/Quadroma (Trion Pharma/Fresenius Biotech), ledge to socket (Genentech), CrossMAbs (Roche), and CH3 electrostatically induced interaction (Chugai, Amgen, NovoNordisk, Oncomed), LUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), Biclonic (Merus) and DuoBody® products (Genmab A/S).

Triomab quadroma technology can be used to generate full length bispecific antibodies. Triomab technology stimulates Fab-arm exchange between two parent chimeric antibodies, one parent mAb having IgG2a and a second parent mAb having rat IgG2b constant regions, to produce chimeric bispecific antibodies.

To create full-length bispecific antibodies, you can use the strategy of the ledge in the cavity. Briefly, selected amino acids that form an interface between CH3 domains in human IgG can be mutated at positions that affect CH3 domain interactions to promote heterodimer formation. A short side chain amino acid (trough) is inserted into the heavy chain of an antibody that specifically binds to the first antigen, and a long side chain amino acid (ledge) is inserted into the heavy chain of an antibody that specifically binds to the second antigen. Upon co-expression of the two antibodies, a heterodimer is formed as a result of the preferential interaction of the pit heavy chain and the ridge heavy chain. Examples of pairs of substitutions in CH3 forming a ridge and a valley are the following (indicated as modified position in the first CH3 domain of the first heavy chain/modified position in the second CH3 domain of the second heavy chain): T366Y/F405A, T366W/F405W, F405W/Y407A, T394W /Y407T, T394S/Y407A, T366W/T394S, F405W/T394S and T366W/T366S L368A Y407V.

CrossMAb technology can be used to generate full length bispecific antibodies of the invention. CrossMAb antibodies, in addition to using the Fab-arm exchange strategy in the protrusion-to-cavity promoter, have an exchange of CH1 and CL domains in one of the halves of the arms to ensure correct pairing of the light chain of the resulting bispecific antibody (see, for example, US patent No. 8242247) .

Other redirection strategies can be used to generate full-length bispecific antibodies by swapping the variable or constant or both between the heavy chain and the light chain, or within the heavy chain of the bispecific antibodies (either in one or both arms). Such exchanges include, for example, exchanges of VH-CH1 with VL-CL, VH with VL, CH3 with CL, and CH3 with CH1, as described in Patent Publication Nos. WO 2009/080252.

Other strategies can be used such as promoting heavy chain heterodimerization using electrostatic interactions by introducing substitutions of positively charged residues on one CH3 surface and negatively charged residues on the other CH3 surface, as described in US Patent Publication No. US 2010/0015133; US Patent Publication No. US 2009/0182127; US Patent Publication No. US 2010/028637 or US Patent Publication No. US 2011/0123532. In other strategies, heterodimerization can be promoted by the following substitutions (indicated modified position in the first CH3 domain of the first heavy chain/modified position in the second CH3 domain of the second heavy chain): L351Y_F405A_Y407V/T394W,

T366I_K392M_T394W/F405A_Y407V, T366L_K392M_T394W/F405A_Y407V, L351Y_Y407A/

T366A_K409F, L351Y_Y407AAT366V_K409F, Y407A/T366A_K409F or T350V_L351Y_F405A_Y407V/

T350V_T366L_K392L_T394W as described in US Patent Publication No. US 2012/0149876 or US Patent Publication No. US 2013/0195849.

To obtain bispecific antibodies, LUZ-Y technology can be used. In this technology, a leucine zipper type sequence is attached to the C-terminus of the CH3 domains to allow heterodimer assembly from the parent mAbs, which is removed after purification.

SEEDbody technology can be used to generate bispecific antibodies. To promote heterodimerization, SEEDbody antibodies in their constant domains have the replacement of selected IgG residues with IgA residues, as described in US Pat. No. US 20070287170.

Bispecific antibodies can be generated in vitro in a cell-free environment by introducing asymmetric mutations in the CH3 region of two monospecific homodimeric antibodies and generating a bispecific heterodimeric antibody from the two original monospecific homodimeric antibodies under reducing conditions to allow disulfide bond isomerization, according to the methods described in an International Patent Publication . No. WO 2011/131746. In these methods, the first monospecific divalent antibody and the second monospecific divalent antibody are designed to have certain substitutions in the CH3 domain that contribute to the stability of the heterodimer; the antibodies are incubated together under reducing conditions sufficient to expose the cysteines in the hinge region to disulfide bond isomerization; thus obtaining a bispecific antibody by exchanging Fab arms. Substitutions F405L in one heavy chain and K409R in the other heavy chain can be used in IgG1 antibodies. In IgG ₄ antibodies, one heavy chain may be wild-type IgG ₄ having F at position 405 and R at position 409, and the other heavy chain may have F405L and R409K substitutions. The incubation conditions can be optimally returned to non-reducing conditions. Examples of reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine and beta-mercaptoethanol. For example, an incubation for at least 90 minutes at a temperature of at least 20° C. in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol at a pH of 5 to 8 can be used, e.g. at pH 7.0 or at pH 7.4.

In some embodiments, the bispecific antibodies comprise recombinant dual-targeting IgG-like molecules, wherein each of the two sides of the molecule contains a Fab fragment or portion of a Fab fragment of at least two different antibodies; fused IgG molecules, in which full-length IgG antibodies are fused with an additional Fab fragment or parts of the Fab fragment; Fc fusion molecules in which single chain Fv molecules or stabilized diabodies are fused to heavy chain constant domains, Fc regions, or portions thereof; fused Fab molecules in which different Fab fragments are fused to each other; heavy chain antibodies based on ScFv and diabodies (e.g., domain antibodies, nanobodies) in which different single chain Fv molecules, or different diabodies, or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other, or to another protein or carrier molecule.

As a rule, substitutions are introduced into the molecule, for example, into the constant domain of an antibody, at the DNA level using standard methods.

The antibodies of the invention can be engineered into a variety of known forms of antibodies.

For example, a bispecific anti-PD-1/CD3 antibody can be generated using the VH/VL domains of the anti-PD-1 antibodies described herein and any of the VH/VL regions of the anti-CD3 antibodies described in the literature.

Another embodiment of the invention is a bispecific antibody containing a first domain that binds to PD-1 and a second domain that binds to CD3.

Methods for characterizing antibodies.

Agonistic antibodies.

An exemplary biological activity induced by the agonistic antibodies provided herein is the inhibition (eg, suppression) of antigen-specific CD4+ or CD8+ T cells. Various indicators can be used to assess the agonism of the antibodies provided herein, such as decreased proliferation or decreased production of interferon-y (IFN-γ), IL-17 IL-2, IL-6, IL-22, IL-23, or GM-CSF with antigen-specific CD4+ or CD8+ T cells. An example assay utilizes the effect of antibodies on normal donor T cells that are stimulated with allogeneic dendritic cells or specific antigens such as tetanus toxoid or CMV. Under such conditions, changes in T cell function upon antibody treatment can be detected by measuring cytokine levels in the supernatant or by activating T cell markers. In the assay example, as a source of antigen-specific CD4+ T cells or

- 35 043064

CD8+ use PBMCs with identified reactivity to CMV antigens. 1.5x10 ⁶ cells/ml or 2x10 ⁶ cells/ml CMV responsive PBMCs are seeded onto culture plates and 0.1-0.2 μg/ml CMV peptides are added to the cultures. CMV peptides are available from, for example, JPT Technologies. Test antibodies are added at a single dose of 10 μg/ml, the plates are incubated for 6 days and cell proliferation is assessed by adding 1 μCi/well of methyl-3H-thymidine (PerkinElmer) for 6 hours and measuring the radioactivity in each sample. Alternatively, cytokine production by cells is measured using ELISA or known multiplex assays.

Antibodies that block the binding of PD-1 to PD-L1 and/or PD-L2.

Antibodies of the invention provided herein (such as agonist antibodies that specifically bind to PD-1) may or may not be ligand-blocking. The ability of agonist antibodies provided herein can be assessed by their ability to block a ligand, eg, PD-L1 and/or PD-L2, using competitive assays such as cell clustering assay.

In an example assay, HEK cells having different labels and overexpressing either PD-1 or PD-L1 (or PD-L2) are mixed in a 1:1 ratio and the ability of antibodies to inhibit clustering of cells expressing PD-1 and PD-L1 is quantified. or PD-1 and PD-L2. Cells overexpressing human PD-1 can be labeled with Violet Cell Trace, and cells overexpressing PD-L1 or PD-L2 can be labeled with Far Red Cell Trace Stain (Life Technologies). Cells expressing PD-1 are mixed with the test antibody, and after a short incubation, cells expressing PD-L1 are added to the mixture. The mixture is incubated for one hour and the percentage of double positive events (eg, cell clusters positive for Violet Cell Trace and Far Rd Cell Trace staining) determined by flow cytometry. The level of clustering is measured by the percentage of double positive events, and the percentage of clustering in the presence of test antibodies is compared with positive and negative isotype control antibodies. An antibody provided herein blocks the binding of PD-L1 (or PD-L2) to PD-1 if the antibody reduces the number of double positive events for cells expressing PD-1 and PD-L1 (or PD-L2) in a statistically significant manner compared to with isotype control, using p<0.01 significance level. An antibody provided herein does not block PD-L1 (or PD-L2) binding to PD-1 if the antibody inhibits PD-1 and PD-L1 (or PD-L2) dual positive events in a non-statistically significant manner, e.g., p >0.01.

Antibody affinity measurements.

The affinity of an antibody for human or non-human PD-1, such as the cynomolgus monkey, can be determined experimentally by any suitable method. Such methods may include the use of ProteOn XPR36, Biacore 3000 or KinExA equipment, ELISA or competitive binding assays known to those skilled in the art. The measured affinity value of a particular antibody/PD-1 interaction may vary when measured under different conditions (eg, osmolarity, pH). Thus, measurements of affinity and other binding parameters (eg, KD, K _on , K _off ) are typically performed under standardized conditions and using a standardized buffer solution, such as the buffer solution described herein in Example 1. Those skilled in the art It will be understood in the art that the intrinsic error of affinity measurements, for example using Biacore 3000 or ProteOn equipment (measured as standard deviation (SD)) can typically be 5-33% for measurements made within typical detection limits. Therefore, the term about in the context of KD characterizes the typical standard deviation in the analysis. For example, a typical CO for a KD value of 1 x 10' ⁹ M is up to +0.33 x 10 ^-9 M.

Antibodies competing for binding to PD-1 with a reference antibody.

The competition of antibodies of the invention containing certain VH and VL sequences (and, for example, reference antibodies) for binding to PD-1 can be analyzed in vitro on the Octet Red384 platform (Forte Bio). Histidine-labeled PD-1 antigen is loaded onto HIS sensors and the sensors are exposed to 20 μg/ml of reference anti-PD-1 antibody followed by exposure to an equal concentration of test anti-PD-1 antibody. Additional binding of test antibody after saturation with reference antibody indicates simultaneous binding of two antibodies to PD-1, and this indicates that reference and test antibody do not compete for binding to PD-1. Alternatively, the absence of additional binding of the test antibody indicates that the two antibodies are competing for binding to PD-1.

Antibodies competing for binding to PD-1 with a reference antibody can be generated by isolating antibodies specifically binding to PD-1 using phage display libraries and screening the resulting antibodies for their ability to compete for binding to PD-1 with the aforementioned reference antibodies.

The antibody under test competes for PD-1 binding with the reference antibody if the test antibody

- 36 043064 the antibody binds to PD-1 at a saturating concentration of the reference antibody. Binding can be detected using biolayer interferometry (eg Octet) by detecting the wavelength shift due to the fact that the bound antibody increases the optical density of the biosensor tip over time.

An antibody epitope.

The PD-1 epitope that binds to an antibody of the invention can be determined, for example, using hydrogen/deuterium exchange (H/D exchange) or by analyzing the crystal structure of the antibody in complex with PD-1. Two anti-PD-1 antibodies bind to the same epitope on PD-1 if about 70% or more of the PD-1 amino acid residues protected by an antibody with at least a 5% difference in H/D deuteration levels are identical for those two antibodies, or if it is determined that 70% or more of the PD-1 amino acid residues associated with the antibody in the crystal structure of the complex of the antibody and PD-1 are identical between the two antibodies. In the crystal structure of an antibody-PD-1 complex, epitope residues are those PD-1 residues that are within 4A or less of any of the antibody CDR residues.

In the H/D exchange assay, the PD-1 protein is incubated in the presence or absence of an antibody in deuterated water for predetermined periods of time, which results in incorporation of deuterium into exchangeable hydrogens not protected by the antibody, followed by digestion of the protein with a protease and analysis of peptide fragments. using liquid chromatography with mass spectrometry (LC-MS). In the assay example, 5 µl of test antibody (10 µg) or 5 µl of PD-1 complex and test antibody (10 and 7.35 µg, respectively) are incubated with 120 µl of deuterium oxide labeling buffer (50 mM phosphate, 100 mM sodium chloride at pH 7.4) for 0 s, 60 s, 300 s, 1800 s, 7200 s and 14400 s. The deuterium exchange reaction is quenched by the addition of 63 μl of 5 M guanidine hydrochloride and the final pH is 2.5. The quenched sample is digested on a pepsin/protease type XIII column and analyzed by LC-MS. For pepsin/protease type XIII digestion, denature 5 µg of samples in 125 µl of control buffer solution (50 mM phosphate, 100 mM sodium chloride at pH 7.4) by adding 63 µl of 5 M guanidine hydrochloride (final pH 2.5) and incubation of the mixture for 3 min. The mixture is subsequently digested with pepsin/protease type XIII and the resulting peptides are analyzed using an UPLC-MS system consisting of a Waters Acquity UPLC chromatograph coupled to a Q Exactive™ Hybrid Quadrupole-Orbitrap (Thermo) mass spectrometer. Raw MS data is processed using HDX Workbench software for analyzing H/D exchange MS data. Deuterium levels are calculated using the average mass difference between the deuterated peptide and its native form (t ₀ ). Peptide identification is performed by searching MS/MS data against the PD-1 sequence using the Mascot software. The mass tolerance for the precursor and product ions is 20 ppm and 0.05 Da, respectively.

For x-ray crystallography, PD-1 and test antibody are expressed and purified using standard protocols. The PD-1/test antibody complex is incubated overnight at 4°C, concentrated and separated from uncomplexed compounds by size exclusion chromatography. The complex is crystallized by vapor diffusion from various known test solutions, for example solutions containing PEG3350, ammonium citrate and 2-(N-morpholino)ethanesulfonic acid (MES).

Antibodies binding the same epitope on PD-1 as reference antibodies can be generated by isolating PD-1 binding antibodies using phage display libraries, selecting those antibodies that compete with the reference antibody for binding to PD-1 on 100%, and identification of the antibody epitope by H/D exchange or by X-ray crystallography.

Alternatively, mice or rabbits can be immunized with peptides containing epitopic residues and the resulting antibodies can be assessed for binding within the indicated region.

Polynucleotides, vectors, host cells.

The invention also provides an isolated polynucleotide encoding any of the antibodies of the invention.

The invention also provides an isolated polynucleotide encoding any of the antibody heavy chain variable regions, any of the antibody light chain variable regions, or any of the antibody heavy chains and/or antibody light chains of the invention.

The invention also provides an isolated polynucleotide encoding a VH of SEQ ID NO: 8, 9, 10, 140, 141, or 142.

The invention also provides an isolated polynucleotide encoding VL with SEQ ID NO: 14, 15, 16, 143 or 144.

The invention also provides an isolated polynucleotide encoding VH of SEQ ID NO: 8, 9, 10, 140, 141, or 142 and VL of SEQ ID NO: 14, 15, 16, 143, or 144.

The invention also provides an isolated polynucleotide encoding a heavy chain (HC) with

- 37 043064

SEQ ID NO: 20, 21, 22, 150, 151 or 152.

The invention also provides an isolated polynucleotide encoding a light chain (LC) with SEQ

ID NO: 26, 27, 28, 153 or 154.

The invention also provides an isolated polynucleotide encoding the HC of SEQ ID NO: 20,

21,22, 150, 151 or 152 and LC with SEQ ID NO: 26, 27, 28, 153 or 154.

The invention also provides an isolated polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 11, 12, 13, 17, 18, 19, 23, 24, 25, 29, 30, 31, 132, 133, 134, 135, 155, 156 , 157, 158, 159, 160, 161, 162, 163 or 164.

The invention also provides an isolated polynucleotide encoding a VH of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, or 51.

The invention also provides an isolated polynucleotide encoding VL with SEQ ID NO: 60, 61 or 62.

The invention also provides an isolated polynucleotide encoding VH of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50 or 51 and VL of SEQ ID NO: 60, 61 or 62.

The invention also provides an isolated polynucleotide encoding the heavy chain of SEQ ID NO: 66, 67, 68, 69, 70, 71, 72, or 73.

The invention also provides an isolated polynucleotide encoding a light chain of SEQ ID NO: 82, 83 or 84.

The invention also provides an isolated polynucleotide encoding HC with SEQ ID NO: 66, 67, 68, 69, 70, 71, 72 or 73 and LC with SEQ ID NO: 82, 83 or 84.

The invention also provides an isolated polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 52, 53, 54, 55, 56, 57, 58, 59, 63, 64, 65, 74, 75, 76, 77, 78, 79, 80 , 81, 85, 86, 87, 136, 137, 138 or 139.

The invention also provides an isolated polynucleotide encoding a VH of SEQ ID NO: 94 or 95.

The invention also provides an isolated polynucleotide encoding VL with SEQ ID NO: 98, 99 or 100.

The invention also provides an isolated polynucleotide encoding VH with SEQ ID NO: 94 or 95 and VL with SEQ ID NO: 98, 99 or 100.

The invention also provides an isolated polynucleotide encoding the HC of SEQ ID NO: 104 or 105.

The invention also provides an isolated polynucleotide encoding an LC with SEQ ID NO: 108, 109 or 110.

The invention also provides an isolated polynucleotide encoding HC with SEQ ID NO: 104 or 105 and LC with SEQ ID NO: 108, 109 or 110.

The invention also provides an isolated polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 96, 97, 101, 102, 103, 106, 107, 111, 112, or 113.

The polynucleotide sequences encoding the VH and/or VL, or antigen-binding fragments thereof, of the antibodies of the invention, or the heavy chain and/or light chain of the antibodies of the invention, may be operably linked to one or more regulatory elements, such as a promoter or enhancer, that allow the expression the nucleotide sequence in the intended host cell. The polynucleotide may be cDNA.

The invention also provides a vector containing a polynucleotide of the invention. Such vectors may be plasmid vectors, viral vectors, baculovirus expression vectors, transposon vectors, or any other vectors suitable for introducing a polynucleotide of the invention into a given organism or genetic environment in any way. For example, polynucleotides encoding the variable regions of the light and/or heavy chains of the antibodies of the invention, optionally linked to constant regions, are inserted into expression vectors. The light and/or heavy chains can be cloned in the same or different expression vectors. The DNA regions encoding VH, VL, HC and/or LC, or antigen-binding fragments thereof, may be operably linked to control sequences in the expression vector(s) that provides(s) for the expression of the polypeptides. Such control sequences include signal sequences, promoters (eg, naturally associated or heterologous promoters), enhancer elements, and transcription termination sequences, and are selected to be compatible with the host cell selected for expression of the antibody. Following the introduction of the vector into the appropriate host, the host is incubated under conditions suitable for high-level expression of the proteins encoded by the introduced polynucleotides.

In some embodiments, the vector contains a polynucleotide of SEQ ID NO: 11 and/or a polynucleotide of SEQ ID NO: 17.

In some embodiments, the vector contains a polynucleotide of SEQ ID NO: 12 and/or a polynucleotide of SEQ ID NO: 18.

In some embodiments, the vector comprises a polynucleotide of SEQ ID NO: 12 and/or a polynucleotide of SEQ ID NO: 19.

In some embodiments, the vector contains a polynucleotide with a linucleotide of SEQ ID NO: 19.

In some embodiments, the vector contains a polynucleotide with a linucleotide of SEQ ID NO: 63.

In some embodiments, the vector contains a polynucleotide with a linucleotide of SEQ ID NO: 64.

In some embodiments, the vector contains a polynucleotide with a linucleotide of SEQ ID NO: 65.

In some embodiments, the vector contains a polynucleotide with a linucleotide of SEQ ID NO: 64.

In some embodiments, the vector contains a polynucleotide with a linucleotide of SEQ ID NO: 64.

In some embodiments, the vector contains a polynucleotide with a linucleotide of SEQ ID NO: 64.

In some embodiments, the vector contains a polynucleotide with a linucleotide of SEQ ID NO: 64.

In some embodiments, the vector contains a polynucleotide with a linucleotide of SEQ ID NO: 64.

In some embodiments, the vector contains a polynucleotide with a linucleotide of SEQ ID NO: 64.

In some embodiments, the vector contains a polynucleotide with a linucleotide of SEQ ID NO: 101.

In some embodiments, the vector contains a polynucleotide with a linucleotide of SEQ ID NO: 102.

In some embodiments, the vector contains a polynucleotide with a linucleotide of SEQ ID NO: 103.

In some embodiments, the implementation of the vector contains a polynucleotide polynucleotide with SEQ ID NO: 133.

In some embodiments, the implementation of the vector contains a polynucleotide polynucleotide with SEQ ID NO: 135.

In some embodiments, the implementation of the vector contains a polynucleotide polynucleotide with SEQ ID NO: 137.

In some embodiments, the implementation of the vector contains a polynucleotide polynucleotide with SEQ ID NO: 139.

In some embodiments, the implementation of the vector contains a polynucleotide polynucleotide with SEQ ID NO: 19.

In some embodiments, the implementation of the vector contains a polynucleotide polynucleotide with SEQ ID NO: 19.

In some embodiments, the implementation of the vector contains a polynucleotide polynucleotide with SEQ ID NO: 19.

SEQ ID NO: 13 and/or SEQ ID NO: 52 and/or SEQ ID NO: 53 and/or SEQ ID NO: 53 and/or SEQ ID NO: 54 and/or SEQ ID NO: 55 and/or SEQ ID NO: 56 and/or SEQ ID NO: 57 and/or SEQ ID NO: 58 and/or SEQ ID NO: 59 and/or SEQ ID NO: 96 and/or SEQ ID NO: 97 and/or SEQ ID NO: 97 and/or pos SEQ ID NO: 132 and/or with SEQ ID NO: 134 and/or with SEQ ID NO: 136 and/or with SEQ ID NO: 138 and/or with SEQ ID NO: 155 and/or with SEQ ID NO: 156 and/or with SEQ ID NO: 157 and/or

In some embodiments, the vector contains a polynucleotide of SEQ ID NO: 13 and/or a polynucleotide of SEQ ID NO: 158.

In some embodiments, the vector contains a polynucleotide of SEQ ID NO: 13 and/or a polynucleotide of SEQ ID NO: 159.

In some embodiments, the polynucleotide vector of SEQ ID NO: 158.

In some embodiments, the polynucleotide vector of SEQ ID NO: 158.

In some embodiments, the polynucleotide vector of SEQ ID NO: 158.

In some embodiments, the polynucleotide vector of SEQ ID NO: 159.

In some embodiments, the polynucleotide vector of SEQ ID NO: 159.

In some embodiments, the polynucleotide vector of SEQ ID NO: 159.

In some embodiments, the polynucleotide vector of SEQ ID NO: 31.

In some embodiments, the polynucleotide vector of SEQ ID NO: 31.

In some embodiments, the vector contains a polynucleotide

SEQID NO:

155 and/or contains a polynucleotide

SEQID NO:

156 and/or contains a polynucleotide

SEQID NO:

157 and/or contains a polynucleotide

SEQID NO:

155 and/or contains a polynucleotide

SEQID NO:

156 and/or contains a polynucleotide

SEQID NO:

157 and/or contains a polynucleotide

SEQID NO:

160 and/or contains a polynucleotide

SEQID NO:

161 and/or contains a polynucleotide

SEQID NO:

162 and/or

- 39 043064 polynucleotide of SEQ ID NO: 31.

In some embodiments, the vector contains a polynucleotide of SEQ ID NO: 25 and/or a polynucleotide of SEQ ID NO: 163.

In some embodiments, the vector contains a polynucleotide of SEQ ID NO: 25 and/or a polynucleotide of SEQ ID NO: 164.

In some embodiments, the polynucleotide vector of SEQ ID NO: 163.

In some embodiments, the polynucleotide vector of SEQ ID NO: 163.

In some embodiments, the polynucleotide vector of SEQ ID NO: 163.

In some embodiments, the polynucleotide vector of SEQ ID NO: 164.

In some embodiments, the polynucleotide vector of SEQ ID NO: 164.

In some embodiments, the polynucleotide vector of SEQ ID NO: 164.

contains a polynucleotide contains a polynucleotide contains a polynucleotide contains a polynucleotide contains a polynucleotide contains a polynucleotide

SEQID NO:

SEQID NO:

SEQID NO:

SEQID NO:

SEQID NO:

SEQID NO:

160 and/or

161 and/or

162 and/or

160 and/or

161 and/or

162 and/or

Generally, suitable expression vectors can replicate in host organisms either as episomes or as an integral part of the host's chromosomal DNA. Typically, expression vectors contain selectable markers, such as ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance, to allow detection of such cells transformed with the desired DNA sequences. The glutamine synthetase system can be used to express recombinant proteins, such as antibodies, in cells.

Suitable promoter and enhancer elements are known in the art. Examples of promoters for expression in a eukaryotic cell include promoter and enhancer elements of immunoglobulin light and/or heavy chain genes; cytomegalovirus immediate-early promoter; herpes simplex virus thymidine kinase promoter; SV40 early and late promoters; a promoter present in the long terminal repeats of the retrovirus; the mouse metallothionein-I promoter; and various tissue-specific promoters known in the art. The choice of an appropriate vector and promoter is well within the level of one of ordinary skill in the art.

Large numbers of acceptable vectors and promoters are known to those skilled in the art; many of them are commercially available for the creation of recombinant constructs. The following vectors are provided as an example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, San Diego, CA, USA); pTrc99A, pKK223-3, pKK233-3, pDR540 and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG and pSVL (Pharmacia), pEE6.1 and pEE14.1 (Lonza).

The invention also provides a host cell containing one or more vectors of the invention. The term host cell refers to the cell into which the vector has been inserted. It should be understood that the term host cell is used to refer not only to a specific claimed cell, but also to the progeny of such a cell, as well as a stable cell line derived from a specific claimed cell. Since some modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not be identical to the original cell, but may also be encompassed by the term host cell as used herein. Such host cells can be eukaryotic, prokaryotic, plant or archaeal cells.

E. coli (Escherichia coli), bacilli such as hay bacillus (Bacillus subtilis), and other enterobacteria such as salmonella, serratia and various types of Pseudomonas are examples of prokaryotic host cells. Other microorganisms such as yeast can also be used for expression. Saccharomycetes (eg S. cerevisiae) and pichias are examples of acceptable yeast host cells. Examples of eukaryotic cells may be mammalian, insect, avian or other animal cells. Mammalian eukaryotic cells include immortalized cell lines such as hybridomas or myeloma cell lines, e.g. mouse cell lines SP2/0 (American Type Culture Collection (ATCC), Manassas, VA, USA, CRL-1581), NS0 cell cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (ATcC CRL-1580). An example of a human myeloma cell line is U266 (ATTC CRL-TIB-196). Other useful cell lines include those derived from Chinese Hamster Ovary (CHO) cells, such as CHO-K1SV (Lonza Biologics, Walkersville, MD, USA), Cho-Κι (ATCC CRL-61) or DG44.

The invention also provides a method for producing an antibody of the invention, comprising culturing a host cell of the invention under antibody expression conditions and isolating the antibody,

- 40 043064 produced by the host cell. Known methods for producing antibodies and their purification. Once synthesized (chemically or recombinantly), whole antibodies, their dimers, single light and/or heavy chains, or other antibody fragments such as VH and/or VL, can be purified according to standard procedures, including ammonium sulfate precipitation, the use of affinity columns, column chromatography, high performance liquid chromatography (HPLC), gel electrophoresis, and the like. (essentially, see Scopes, Protein Purification (Springer-Verlag, N.Y., 1982)). The claimed antibody may be substantially pure, for example, at least about 80 to 85% pure, at least about 85 to 90% pure, at least about 90 to 95% pure, at least from about 98 to 99% or more, for example free of contaminants such as cell debris, macromolecules, etc., other than the claimed antibody.

The invention also provides a method for producing an antibody specifically binding to PD-1, comprising combining in an expression vector a first polynucleotide encoding the VH of the antibody and a second polynucleotide encoding the VL of the antibody;

transformation of the host cell with this expression vector;

culturing the host cell in a culture medium under conditions in which VL and VH are expressed and an antibody is formed; and isolating the antibody from the host cell or culture medium.

Polynucleotides encoding certain VH or VL sequences of the invention can be incorporated into vectors using standard molecular biology techniques. Transformation of the host cell, culture, expression and purification of the antibody are performed using well known methods.

Pharmaceutical Compositions/Introduction.

The present invention also provides pharmaceutical compositions comprising the antibodies or antigen-binding fragment thereof of the invention and a pharmaceutically acceptable carrier. For therapeutic use, it is possible to obtain antibodies according to the invention in the form of pharmaceutical compositions containing an effective amount of antibodies as an active ingredient in a pharmaceutically acceptable carrier. The term carrier refers to a diluent, adjuvant, excipient, or carrier vehicle with which an antibody of the invention is administered. Such carrier media may be liquids such as water and oils, including oils of mineral, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. For example, 0.4 % saline solution and 0.3% glycine solution. These solutions are sterile and essentially free of particulate matter. They can be sterilized using well known standard sterilization techniques (eg filtration). The compositions may contain pharmaceutically acceptable excipients necessary to approach physiological conditions, such as pH adjusting and buffering agents, stabilizing, thickening, wetting and coloring agents, etc. The concentration of antibodies or their antigen-binding fragments according to the invention in such a pharmaceutical composition may vary from less than about 0.5%, typically at least about 1%, and up to 15 or 20 wt.%, and can be selected mainly based on the required dose, fluid volumes, viscosity values, etc. according to the specific route of administration chosen. Suitable carrier media and formulations including other human proteins, such as human serum albumin, are described, for example, in Remington: The Science and Practice of Pharmacy, ^21st Edition, Troy, DB ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, see especially pp. 958-989.

The route of administration for therapeutic use of the antibodies or antigen-binding fragments thereof of the invention may be any acceptable route of delivery of the antibody to a patient, such as parenteral administration, e.g. intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, intranasal, intravaginal, rectal), in the form of a composition in a tablet, capsule, solution, powder, gel, particle; and administering the antibody contained in a syringe, implant device, osmotic pump, cartridge, micropump, or other means well known in the art and obvious to the skilled artisan. Localized administration can be achieved, for example, by delivery to a tumor, joint, bronchi, abdominal cavity, capsule, cartilage, cavity, cerebellum, cerebral ventricle, colon, cervix, stomach, liver, myocardium, bone, pelvis, pericardium, cavity abdomen, pleura, prostate, lungs, rectum, kidney, retina, spine, bursa, thorax, uterus, vessel, inside the bladder, damaged tissue, vaginally, rectally, buccally, sublingually, intranasally or transdermally.

The antibodies or antigen-binding fragments thereof of the invention may also be administered prophylactically to reduce the risk of developing an autoimmune disease and/or delay the onset of symptoms.

The antibodies or antigen-binding fragments of these antibodies of the invention may be lyophilized for storage and reconstituted in a suitable vehicle prior to use. This method has been proven to be effective for standard protein preparations, and well known lyophilization and reconstitution techniques can also be used with it.

Methods and applications.

The antibodies or antigen-binding fragments thereof according to the invention are used in in vitro and in vivo diagnostics, as well as for treatment and prevention. For example, the antibodies or antigen-binding fragments thereof of the invention may be administered to cells in in vitro or ex vivo culture or to a patient for the purposes of treating, preventing and/or diagnosing various diseases such as an immune disorder or any condition in which a reduction in T activity is desirable. -cells expressing PD-1, and/or reduced immune response.

The invention also provides a method for suppressing the activation of a PD-1 expressing T cell in a patient, comprising administering to the patient an isolated antibody or an antigen-binding fragment thereof according to the invention for a time period sufficient to suppress the activation of a PD-1 expressing T cell.

In some embodiments, the PD-1 expressing T cell is an antigen-specific CD4+ T cell.

In some embodiments, the PD-1 expressing T cell is a CD8+ antigen specific T cell.

The term "inhibit activation" refers to the ability of the antibodies provided herein to inhibit the activation of PD-1 expressing T cells, such as inhibiting the proliferation or production of IFN-γ by antigen-specific CD4+ and/or CD8+ T cells. An antibody inhibits the activation of PD-1 expressing T cells if the antibody inhibits the proliferation or production of IFN-γ by antigen-specific CD4+ and/or CD8+ T cells by 20, 30, 40, 45, 50, 55, 60, 65, 70, 75%, 80%, 85%, 90%, 95%, or 100% greater than in the absence of antibody (eg, compared to a negative control), or if inhibition is statistically significant compared to inhibition in the absence of antibody.

A PD-1 expressing T cell may be located in close proximity to the site of an inappropriate inflammatory response. The antibodies or antigen-binding fragments thereof of the invention can suppress the activation of PD-1 expressing T cells by enhancing downstream PD-1 signaling, resulting in inhibition of TCR signaling and inhibition of T cell activation, proliferation and/or survival. In an alternative embodiment, the antibodies of the invention can mediate the killing of PD-1 positive T cells through the antibody-mediated ADCC, ADCP and/or CDC effector functions.

Also provided is a method for reducing an immune response, comprising administering to a patient in need thereof a therapeutically effective amount of an isolated antibody or antigen-binding fragment thereof according to the invention to reduce the immune response.

Also provided is a method for treating an immune disorder, comprising administering to a patient in need thereof a therapeutically effective amount of an isolated antibody or antigen-binding fragment thereof according to the invention for the treatment of an immune disorder.

The immune disorder may be chronic or acute, such as a chronic inflammatory disease or an acute inflammatory disease.

In some embodiments, the immune disorder is arthritis, rheumatoid arthritis, asthma, chronic obstructive pulmonary disease (COPD), pelvic inflammatory disease, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, psoriatic arthritis, vasculitis, surgical adhesions, stroke, type I diabetes, Lyme disease, meningoencephalitis, autoimmune uveitis, multiple sclerosis, lupus (such as systemic lupus erythematosus), Guillain-Barré syndrome, atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Graves' disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's granulomatosis, other autoimmune disorders, pancreatitis, trauma (surgery), graft versus host disease, rejection transplant, heart disease, including ischemic diseases such as myocardial infarction, as well as atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis and hypochlorhydria, infertility associated with maternal fetal intolerance, Sjögren's syndrome, vitiligo, myasthenia gravis or systemic scleroderma.

In some embodiments, the immune disorder is rheumatoid arthritis.

In some embodiments, the immune disorder is graft versus host disease.

In some embodiments, the immune disorder is arthritis.

In some embodiments, the immune disorder is asthma.

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In some embodiments, the disease or disorder is COPD.

In some embodiments, the immune disorder is a pelvic inflammatory disease.

In some embodiments, the immune disorder is Alzheimer's disease.

In some embodiments, the immune disorder is an inflammatory bowel disease.

In some embodiments, the immune disorder is Crohn's disease.

In some embodiments, the disease or disorder is ulcerative colitis.

In some embodiments, the immune disorder is Peyronie's disease.

In some embodiments, the immune disorder is celiac disease.

In some embodiments, the implementation of the immune disorder is a disease of the gallbladder.

In some embodiments, the immune disorder is a pilonidal disease.

In some embodiments, the immune disorder is peritonitis.

In some embodiments, the immune disorder is psoriasis.

In some embodiments, the immune disorder is psoriatic arthritis.

In some embodiments, the implementation of the immune disorder is a vasculitis.

In some embodiments, the immune disorder is a surgical adhesion.

In some embodiments, the immune disorder is a stroke.

In some embodiments, the immune disorder is type I diabetes.

In some embodiments, the immune disorder is Lyme disease.

In some embodiments, the immune disorder is meningoencephalitis.

In some embodiments, the implementation of the immune disorder is an autoimmune uveitis.

In some embodiments, the immune disorder is multiple sclerosis.

In some embodiments, the immune disorder is lupus.

In some embodiments, the immune disorder is systemic lupus erythematosus.

In some embodiments, the immune disorder is Guillain-Barré syndrome.

In some embodiments, the immune disorder is atopic dermatitis.

In some embodiments, the immune disorder is autoimmune hepatitis.

In some embodiments, the immune disorder is fibrosing alveolitis.

In some embodiments, the immune disorder is Graves' disease.

In some embodiments, the implementation of the immune disorder is due to IgA nephropathy.

In some embodiments, the immune disorder is idiopathic thrombocytopenic purpura.

In some embodiments, the immune disorder is Meniere's disease.

In some embodiments, the immune disorder is pemphigus.

In some embodiments, the immune disorder is primary biliary cirrhosis.

In some embodiments, the immune disorder is sarcoidosis.

In some embodiments, the immune disorder is scleroderma.

In some embodiments, the immune disorder is Wegener's granulomatosis.

In some embodiments, the immune disorder is pancreatitis.

In some embodiments, the immune disorder is transplant rejection.

In some embodiments, the implementation of the immune disorder is a heart disease, including ischemic diseases such as myocardial infarction.

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In some embodiments, the immune disorder is atherosclerosis.

In some embodiments, the implementation of the immune disorder is an intravascular coagulation.

In some embodiments, the immune disorder is bone resorption.

In some embodiments, the immune disorder is osteoporosis.

In some embodiments, the immune disorder is osteoarthritis.

In some embodiments, the immune disorder is periodontitis.

In some embodiments, the immune disorder is hypochlorhydria.

In some embodiments, the immune disorder is Sjögren's syndrome.

In some embodiments, the immune disorder is vitiligo.

In some embodiments, the immune disorder is myasthenia gravis.

In some embodiments, the immune disorder is systemic scleroderma.

Also provided is a method for treating pain associated with inflammation, comprising administering to a patient in need thereof a therapeutically effective amount of an isolated antibody or antigen-binding fragment thereof according to the invention for treating pain associated with inflammation.

The invention also provides an antibody of the invention that specifically binds to PD-1 for use in therapy.

The invention also provides an antibody of the invention that specifically binds to PD-1 for the treatment of an immune disorder.

The invention also provides an antibody of the invention that specifically binds to PD-1 for the treatment of rheumatoid arthritis.

The invention also provides an antibody of the invention that specifically binds to PD-1 for the treatment of lupus, such as systemic lupus erythematosus.

The invention also provides an antibody of the invention that specifically binds to PD-1 for the treatment of graft versus host disease.

The invention also provides the use of an antibody of the invention that specifically binds to PD-1 in the development of a medicament for the treatment or prevention of an immune disorder.

Combined therapies.

The antibodies provided herein may be administered in combination with a second therapeutic agent.

The second therapeutic agent may be any known treatment for autoimmune disorders such as autoimmune or inflammatory diseases, including any agent or combination of agents known to be useful or that has been or is being used to treat these diseases. Such therapies and therapeutic agents include surgery or surgical procedures (eg, splenectomy, lymphadenectomy, thyroidectomy, plasmapheresis, leukophoresis, cell, tissue, or organ transplantation, bowel procedures, organ perfusion, and the like), radiation therapy, such therapy, such as steroid and non-steroid therapy, hormonal therapy, cytokine therapy, therapy with dermatological agents (eg, topical agents used to treat skin conditions such as allergies, contact dermatitis, and psoriasis), immunosuppressive therapy, and therapy with other anti-inflammatory monoclonal antibodies.

The second therapeutic agent may be a corticosteroid, an antimalarial drug, an immunosuppressant, a cytotoxic drug, or a B cell modulator.

In some embodiments, the second therapeutic agent is prednisone, prednisolone, methylprednisolone, deflascort, hydroxychloroquine, azathioprine, methotrexate, cyclophosphamide, mycophenolate mofetil (MMF), mycophenolate sodium, cyclosporine, leflunomide, tacrolimus, RITUXAN® (rituximab), or BENLYSTA® (belimumab). ).

In some embodiments, the antibodies of the invention are administered in combination with a second therapeutic agent. Examples of second therapeutic agents include corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), salicylates, hydroxychloroquine, sulfasalazine, cytotoxic drugs, immunosuppressive drugs, immunomodulating antibodies, methotrexate, cyclophosphamide, mizoribine, chlorambucil, cyclosporine, tacrolimus (FK506; ProGrafrM), mycophenolate mofetil and azathioprine (6-mercaptopurine), sirolimus (rapamycin), deoxyspergualine, leflunomide and its malononitriloamide analogues; anti-CTLA4 antibodies and fusion Igs, antibodies to B-lymphocyte stimulators (e.g. LYMPHOSTAT-BTM) and CTLA4-Ig fusion proteins (BLyS-44 043064

Ig), CD80 antibodies, anti-T cell antibodies such as CD3 antibodies (OKT3), CD4 antibodies, corticosteroids such as, for example, clobetasol, halobetasol, hydrocortisone, triamcinolone, betamethasone, fluocinol, fluocinonide, prednisone, prednisolone, methylprednisolone; non-steroidal anti-inflammatory drugs (NSAIDs), such as, for example, sulfasalazine, medicines containing mesalamine (known as 5-ASA agents), celecoxib, diclofenac, etodolac, fenprofen, flurbiprofen, ibuprofen, ketoprofen, meclofamate, meloxicam, nabumetone, naproxen, oxaprozin , piroxicam, rofecoxib, salicylates, sulindac and tolmetin; phosphodiesterase-4 inhibitors, anti-TNFa antibodies REMICADE® (infliximab), SIMPONI® (golimumab) and HUMIRA® (adalimumab), thalidomide or analogues such as lenalidomide.

The antibodies of the invention may be administered in combination with a second therapeutic agent simultaneously, sequentially or separately.

Treatment efficacy for rheumatoid arthritis can be assessed using efficacy as measured by clinical responses as defined by the American College of Rheumatology criteria, the European League Against Rheumatism criteria, or any other criteria. See, for example, Felson et al. (1995) Arthritis Rheum. 38:727-35 and van Gestel et al. (1996) Arthritis Rheum. 39:34-40.

Although the invention has been described in general terms, embodiments of the invention will be further described in the following examples, which should not be construed as limiting the scope of the claims.

Example 1 Methods.

Affinity measurements by surface plasmon resonance (SPR).

Affinity measurements were performed using the ProteOn XPR36 system.

(BioRad). A biosensor surface was prepared by binding an anti-human IgG Fc antibody (Jackson cat. no. 109-005-098) to an alginate polymer layer-modified surface of a GLC chip (BioRad, cat. no. 176-5011) using the manufacturer's instructions for amine coupling chemistry. Approximately 5000 mAb response units (RU) were immobilized. Kinetic experiments were performed at 25° C. in running buffer solution (DPBS+0.01% polysorbate P20+100 μg/ml BSA). For kinetic experiments, 200 RU mAb was immobilized followed by administration of analytes (human and cynomolgus PD-1) at concentrations ranging from 1.563 nM to 400 nM (4-fold serial dilutions). The association phase was monitored for 3 minutes at 50 μl/min followed by a 10 or 15 minute buffer flow (dissociation phase). The chip surface was regenerated with two 18 s pulses of 100 mM H ₃ PO ₄ (Sigma cat. no. 7961) at 100 μl/min.

The obtained data were processed using the ProteOn Manager software. First, the data was corrected against the background using intermediate plots. Subsequently, a double subtraction of the data standard was performed by injecting a buffer solution instead of injecting analytes. Kinetic data analysis was performed using a 1:1 Langmuir binding model. The result for each mAb was recorded in the format of K _a (rate of association), K _d (rate of dissociation) and K _D (equilibrium dissociation constant).

Inhibition of antigen-specific T cells. Analysis of cytomegalovirus (CMV)-induced activation of peripheral blood mononuclear cells (PBMC) (CMV-PBMC).

A specific secondary response to CMV assay was used to assess the ability of the generated antibodies to inhibit T cell activation, as measured by inhibition of cell proliferation when CMV responsive donor PBMCs were treated with a mixture of CMV peptides (JPT Technologies).

According to the respective vendors, PBMCs (Astarte Biologics, Hemacare, Precision for Medicine) were responsive to CMV antigens. Frozen PBMC vials were purchased from suppliers and stored in liquid nitrogen. On the day of the experiment, an aliquot of frozen PBMCs was thawed and cells were resuspended in 10 ml assay medium (RPMI-1640/Glutamax/HEPES containing 1% penicillin/streptomycin, 1% sodium pyruvate, 1% non-essential amino acids (NEAA), 10% heat-inactivated fetal calf serum (all reagents purchased from Thermo Fisher Scientific) Cells were centrifuged at 200 g for 15 minutes at room temperature After centrifugation, the supernatant was removed, cells were resuspended in 10 ml of assay medium and centrifuged at 250 g for 10 minutes After centrifugation cells were resuspended in 10 ml assay medium, passed through a 70 µm filter and counted.Cell concentration was adjusted to 1.5 x 106 cells/ml or 2 x 106 cells/ml and cells were plated at 100 µl/well in tissue culture-treated 96-well round bottom plates ( Corning) Cell density for seeding was donor-specific and was determined in preliminary experiments to maximize the analysis window. The mixture of CMV peptides was prepared according to the manufacturer's instructions. Briefly, 40 μl of dimethyl sulfoxide (DMSO) (Sigma) was added to a vial containing 25 μg of lyophilized CMV peptide mixture powder and carefully pipetted to dissolve the reagent. Mixture mother liquor

- 45 043064 CMV peptides in DMSO were diluted in phosphate-buffered saline (PBS) to obtain a solution of 50 μg/ml and left at room temperature for 10 minutes. An additional dilution was performed with the assay medium to four times the final concentration and 50 μl of the CMV peptide solution was added to each stimulated well; and unstimulated control wells were simultaneously injected with medium without additives. The final concentration was optimized for certain donor PBMCs in preliminary experiments (0.1-0.2 µg/ml). Anti-PD-1 antibody was added at a single dose of 10 µg/mL, serially diluted with assay medium to four times the final concentration, and 50 µL of diluted antibody solution was added to each well, while 50 µL of medium was added to control wells without antibody. The plates were incubated at 37°C and 5% CO ₂ for 6 days. After incubation, 100 µl of the supernatant was removed from each well and 100 µl of assay medium containing 1 µCi/well of methyl-3H-thymidine (PerkinElmer) was added to each well and the plates were incubated for 6 h at 37°C and 5% CO2 . Cells were harvested onto Unifilter-96, GF/C plates (PerkinElmer) which were allowed to dry overnight at room temperature. Fifty µl of Microscint-20 reagent (PerkinElmer) was added to each well. The plates were sealed and counted using a TopCount NXT instrument (PerkinElmer).

Inhibition of cell clustering to determine the ability of antibodies to block ligand-receptor interaction.

To assess the ability of the generated antibodies to inhibit the interaction of the receptor and the ligand in the cellular context, which was quantified by the inhibition of cell clustering by flow cytometry, a 1:1 mixture of human embryonic kidney cells (HEK) with overexpression of PD-1 (PD-1-HEK cells) was analyzed. ), or PD-1 ligand PD-L1 (PD-L1-HEK), or PD-L2 (PD-L2-HEK cells).

HEK cells overexpressing human PD1 (Crown Bio) were labeled with Violet Cell Trace (Life Technologies). HEK cells overexpressing cynomolgus monkey PD-L1 or PD-L2 were labeled with Far Red Cell Trace Stain (Life technologies). Briefly, the dyes were solubilized in 20 µl DMSO to give a 5 mM solution, then 5 µl was diluted in 10 ml PBS to make a 2.5 µM solution. Cells were counted and 50x106 cells were centrifuged at 1000 rpm for 5 min at room temperature. Cells were washed once in PBS and 1x106 cells were left as unstained controls. After re-centrifugation, the supernatant was removed and the cells were resuspended in the appropriate dye solutions described above and incubated for 15 min at room temperature. Then 4 ml of ice-cold fetal calf serum (FBS) was added and incubated for another 5 minutes. Cells were then centrifuged as described above, washed once in assay buffer (PBS, 10% FBS, 1 mM EDTA), centrifuged again, supernatant aspirated, then each cell type was resuspended in assay buffer at 3x106 cells/mL. Test antibodies were diluted three times to the final desired concentration (60 μg/ml) in assay buffer. To obtain final cell/antibody samples (in triplicate), 100 μl of PD-1-HEK cells were mixed with 100 μl of antibody. After a 10 minute incubation, 100 μl PDL1-HEK or PD-L2-HEK cells were added and the final mixed samples were incubated on ice for at least one hour. Finally, 5 μl of propidium iodide was added and the samples were gently mixed, transferred to polystyrene round bottom tubes and analyzed on an LSRII flow cytometer (BD Biosciences). After live cell events were gated, the percentage of double positive events for Pacific Blue and APC channels was determined using Flowjo software and plotted in GraphPad Prism 6. The level of clustering measured by the percentage of double positive events for anti-PD-1 mAb was compared with positive events. and negative isotype control antibodies.

Epitope groups according to Octet.

Epitope grouping of antibodies by competitive binding assay was performed on the Octet Red384 platform (Forte Bio) based on biolayer interferometry. This technique measures the binding of a parent antibody to a PD-1 coated biosensor by the wavelength shift caused by the bound antibody increasing the absorbance of the biosensor tip over time. Briefly, histidine-labeled PD-1 antigen was applied to HIS sensors. The sensors were then exposed to 20 μg/ml of the primary anti-PD-1 antibody followed by an equal concentration of the second anti-PD-1 antibody. Data were processed using ForteBio Data Analysis software. Additional binding to the second antibody after saturation with the first antibody indicates simultaneous binding of the two antibodies, which necessarily means unique, non-overlapping epitopes. Alternatively, the absence of additional binding indicates that the two antibodies are competing for binding to the PD-1 antigen.

Antibody-dependent cellular cytotoxicity (ADCC).

Activation of human memory T cells (target cells).

Frozen aliquots of human memory T cells (AllCells LLC) were thawed in a water bath at 37°C and resuspended in 40 ml culture medium (RPMI+10% FBS or 5% HuSAB+50

- 46 043064 μM βΜΕ (1:1000) + 1% GlutaMax + 10 mM Hepes. Cells were centrifuged at 250 g for 15 min at room temperature. After centrifugation, the supernatant was removed, the cells were resuspended in 10 ml of the culture medium and centrifuged at 250 g for 10 min. After centrifugation, the cells were resuspended in 10 ml of assay medium and counted. The cell concentration was adjusted to 1.0 x 10 ⁶ cells/ml and 10 ml of the cell suspension was transferred to tissue culture plates pre-coated with anti-CD3 antibody (eBioscience, 5 μg/ml in PBS, 1 h at 37°C). The culture medium was supplemented with 2 μg/ml of anti-CD28 antibody (eBioscience) and a cocktail of cytokines IL-2, IL-15, and IL-7 (R&D systems and Peprotech) at a concentration of 100 ng/ml. Preliminary experiments indicated that PD-1 expression peaked on day 5 post-activation and hence this time point was chosen for analysis. Freshly isolated resting memory T cells (not activated by CD3/CD28 stimulation) were also used as controls, as they express low levels of PD-1.

Obtaining effector cells (NK92.CD16 and PBMC).

NK-92 cells were grown in tissue culture flasks, which were stored upright, at a density of 0.5-1.0x10 ⁶ cells/ml in 40 ml of medium (Myelocult H5100 (StemCell Technologies), 1X sodium pyruvate/minor amino acids/Pen Strep (Invitrogen), 4 μM hydrocortisone (StemCell Technologies), 100 ng/ml rhIL-2 (R&D Systems)). Frozen PBMC cells (Hemacare) were thawed the day before the experiment and resuspended in XVIVO-10 medium (Lonza), 10% HI FBS (Invitrogen) and 100 ng/ml IL2 (R&D Systems). Cells were centrifuged at 250 g for 15 min at room temperature. After centrifugation, the supernatant was removed, the cells were resuspended in 10 ml of the culture medium and centrifuged at 250 g for 10 min. After centrifugation, cells were resuspended in 10 ml of culture medium and counted. The cell concentration was adjusted to 1.0 x 10 ⁶ cells/ml and the required number of cells was applied to a tissue culture dish (TC).

On the day of analysis, PBMC and NK cells were harvested and resuspended at 1 x 10 ⁷ cells/ml and 4 x 10 ⁶ cells/ml, respectively, in assay medium (RPMI, 10% FBS, 1 mM sodium pyruvate, 0.1 mM NEAA). Memory T cells were washed once, resuspended at 1×10 ⁶ cells/ml and labeled with 6 µl BATDA (Perkin Elmer) per ml cells at 37° C. for 30 minutes. The BATDA-labeled cells were washed three times with excess cold assay medium and adjusted to a density of 0.2 x 10 ⁶ cells/ml. Serial dilutions of test mAbs were prepared, starting at 20 μg/ml, in assay medium (100 μl, 2x final concentration) in 96-well U-bottom assay plates. BATDA labeled target cells (50 μl) were added at 0.2 x 10 ⁶ cells/ml and incubated with test mAbs. PBMCs (50 µl) were added at a concentration of 1 x 10 ⁷ cells/ml to give a ratio of 50:1 effector cells:target cells, while NK cells (50 µl) were added at a concentration of 4 x 10 ⁶ cells/ml to give a ratio of 20:1 effector cells. cells: target cells. Control wells with maximum lysis containing labeled target cells and 20 μl of 2% Triton X-100 were prepared in triplicate. Control wells with minimal lysis (background release of BATDA) containing labeled target cells in assay medium were also prepared in triplicate. The final volume in all wells of the assay plate was 200 μl. The contents of the wells were carefully mixed with a pipette. The plates were centrifuged for a short time and incubated at 37° C. in a 5% CO ₂ incubator for 2.5 hours. After incubation, the cells were centrifuged at 1200 rpm for 5 minutes. Supernatants (30 μl) were transferred to 96-well white opaque Nunc plates (ThermoFisher, 136101) containing 200 μl of europium solution (PerkinElmer, C135-100). The plates were closed to protect from light and mixed for 15 min on a shaker. Samples were read on an Envision MultiLabel Reader (PerkinElmer). The percentage of lysis was calculated as follows: 100x (experimental release - background release)/(maximum release - background release).

Complement dependent cytotoxicity (CDC).

Frozen aliquots of human total T cells (Biological Specialty, LSII 49301C) were thawed in a water bath at 37°C and resuspended in 40 ml of prewarmed RPMI 1640+Glutamax+25 mM HEPES medium (Life Technologies, cat. no. 72400-047)+ 10% FBS (Gibco, 160000-36). Cells were centrifuged at 250 g for 5 min at room temperature. After centrifugation, the supernatant was removed, the cells were resuspended in 10-20 ml of the medium and counted. Human total T cells (Biological Specialty, LSII 49301C) were activated for 5-6 days prior to CDC analysis with Human T-Activator CD3/CD28 Dynabeads™ (Life Technologies, cat. no. 11132D). Briefly, 75 µl of pre-washed Dynabeads was mixed with T cells 6.0 x 10 ⁶ cells/vial, added to T175 in 10-20 ml medium and incubated for 6 days at 37°C in a 5% CO2 incubator. After 6 days, the pellets were removed from the mixture using EasySep™ magnets (STEMCELL Technologies, 18000).

In preparation for CDC assays, cells were centrifuged at 250 g for 5 min at room temperature. After centrifugation, the supernatant was removed, the cells were resuspended in 10 ml of serum-free culture medium and counted. The cell concentration was adjusted to 1.6x10 ⁶ cells/ml in serum-free medium and seeded at 50 μl/well in 96-well U-bottom plates.

- 47 043064 (Falcon, 353077). The test antibodies were serially diluted eleven times in a ratio of 1:3 in a serum-free medium, starting at a concentration of 25 μg/ml (three times). Test antibodies were added to the appropriate wells with target cells at 50 μl/well. Serum-free medium was added to the wells for background and lysis control at 50 μl/well. The plates were closed and incubated for 1 hour at room temperature (room temp.). 50 μl/well of 10% (three times) rabbit complement (Invitrogen, 31038-100) diluted in serum-free medium was added to test wells. Serum-free medium was added to the background wells at 50 μl/well. 2% Triton-X in serum-free medium was added to lysis control wells at 50 μl/well. The plates were incubated at 37°C and 5% CO ₂ for 60 min. Cells were centrifuged at 250 g for 5 min. Remove 50 μl of the supernatant from the well and transfer to 96-well flat-bottomed plates for UV-Vis spectroscopy (Corning, 3635). 50 µl/well of lactate dehydrogenase (LDG) detection reagent (Roche, 11-644-793-001) was added to each sample; the plates were closed and incubated for 15 min at room temperature. Absorbance was determined at 490 nm and 650 nm on a SpectraMax Plus M5 spectrophotometer (Molecular Devices). Statistical analysis was performed using Microsoft Excel and GraphPad Prism 6. The absorbance at 650 nm was subtracted from the value at 490 nm to normalize for haze. Percentage (%) cytotoxicity was determined for each sample using the following formula:

(experimental-low control)/(high control-low control)x100, where high control is the average of the Triton-X lysis control wells and low control is the average of the background control wells (medium only). A four parameter logistic curve fit model, [log(agonist) vs. response - variable slope (four parameters)], was used in GraphPad Prism to plot the log10 antibody concentration versus calculated % cytotoxicity. Samples were analyzed in duplicate and the analysis was performed in duplicate.

To confirm target expression, PD-1 levels on activated human total T cells were measured by flow cytometry on day 5 or 6 post-activation. Briefly, T cells were centrifuged at 250 g for 5 min and resuspended at 1x106 cells/ml in BSA Stain Buffer (BD Biosciences, 554657). 100-200 thousand cells were incubated in 100 μl of total volume with saturating concentrations of antibodies to PE-PD-1 (Biolegend, 329906). Cells were washed twice with BSA Stain Buffer and resuspended in an equal volume of buffer containing DRAQ7 live/dead dye (Cell Signaling Technology, 7406S). Median fluorescence intensity was recorded for 5,000 live cell events on a MACSQuant Analyzer 10 flow cytometer. Receptor levels were determined using a standard curve generated with Quantibrite™ PE Beads (BD, 340495) and expressed as the number of bound antibodies per cell.

Example 2 Preparation of agonist antibodies that specifically bind to PD-1 and their structural characterization.

Three Balb/c mice and three C3H mice were immunized with the extracellular domain (ECD) of human PD-1 (SEQ ID NO: 1) conjugated to Fc (huPD-1-Fc) and hybridomas were generated using standard protocols. Hybridomas were screened by ELISA for binding to recombinant PD-1 (ECD). Hits were considered samples that gave an ELISA signal more than five times higher than the average value of the negative control. Positive clones were cross-screened for irrelevant Fc fusion protein and for binding to mouse PD-1. Supernatants from cloned single cell hybridomas were tested for binding to human and cynomolgus monkey PD-1 protein. Hits were considered a signal exceeding the average of the negative controls+3 standard. off

Selected mouse antibodies were cloned into human IgG1 as chimeric mAbs and tested for the ability to inhibit antigen-specific T cell activation in a CMV secondary response assay according to the protocol described in Example 1 (CMV-PBMC assay). In FIG. 1A and 1B show that most of the antibodies tested inhibited T cell proliferation at levels above 50% or more. PD1B199 is a PD-1 antagonist mAb. CNTO3930: isotype control.

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SEQ ID NO: 1 PD-1 ECD

PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQ

TDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQI

KESLRAELRVTERRAEVPTAHPSSPSPRPAGQFQTLV

SEQ ID NO: 131 FL mature PD-1

PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQ TDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQI KESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICS RAARGTIGARRTGQPLKEDPSAVPVFS VDYGELDFQWREKTPEPPVPCVPEQTEYATIVF PSGMGTSSPARRGSAADGPRSAQPLRPEDGHCSWPL

Example 3 Humanization of Anti-PD-1 Antibodies.

Several parent antibodies, including PD1B505, PD1B506, and PD1B512, were humanized. To find the best combination of humanized VH and VL, one or more human germline heavy and light chain V region sequences were selected for each of the antibodies. Human J-segments for the VL and VH of each parent antibody were selected by comparing the original J-segment sequence with human J-segment sequences to maximize sequence identity. All humanized VH/VL pairs of each antibody were made and tested in matrix as crude supernatants for antigen binding and protein expression. Based on these data, antibodies showing comparable or higher binding to PD-1 than the parent mouse antibody were purified and tested for efficacy in the CMV-PBMC assay.

The resulting antibodies were analyzed for possible unwanted risks of post-translational modification. High-risk deamidation motifs located in the CDRs and free cysteines anywhere in the antibody were removed by mutagenesis and the resulting antibodies were tested for their binding to PD-1 and efficacy in the CMV-PBMC assay.

Humanized antibodies and their variants were cloned as IgG1.

In table. 3 shows the generated antibodies. In table. 4 shows the SEQ ID NOs for the amino acid sequences of the VH, VL, HC, and LC regions of antibodies. In table. 5 shows SEQ ID NOs for polynucleotide sequences encoding the VH, VL, HC, and LC regions of antibodies. In table. 6 shows the SEQ ID NOs for the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 antibody regions. In table. 7 shows the amino acid sequences of the HCDR1, HCDR2 and HCDR3 antibody regions. In table. 8 shows the amino acid sequences of the LCDR1, LCDR2, and LCDR3 regions of antibodies. In table. 9 shows the amino acid sequences of the VH and VL regions of antibodies. In table. 10 shows polynucleotide sequences encoding the VH regions of antibodies. In table. 11 shows polynucleotide sequences encoding the VL regions of antibodies. In table. 12 shows the amino acid sequences of the HC region. In table. 13 shows the amino acid sequences of the LC region. In table. 14 shows the polynucleotide sequences encoding the HC regions of antibodies. In table. 15 shows polynucleotide sequences encoding the LC regions of antibodies.

Table 3

wal Origin of the antibody Title VH Title VL PD1B505 Initial PD1H93 PD1L30 PD1B742 Humanized PD1B505 PD1H384 PD1L468 PD1B743 Humanized PD1B505 PD1H384 PD1L469 PD1B878 C848-variant of PD1B743 PD1H405 PD1L469 PD1B506 Initial PD1H90 PD1L28 PD1B750 Humanized PD1B506 PD1H388 PD1L470 PD1B751 Humanized PD1B506 PD1H388 PD1L471 PD1B845 C56A-variant PD1B750 PD1H399 PD1L470 PD1B846 N55D, O56A-variant RP1V750 PD1H400 PD1L470 PD1B847 N55Q-BapHaHT PD1B750 PD1H401 PD1L470 PD1B848 M55K-variant of PD1B750 PD1H402 PD1L470 PD1B849 M55E-variant of PD1B750 PD1H403 PD1L470 PD1B85O O561-variant PD1B750 PD1H404 PD1L470 PD1B512 Initial PD1H81 PD1L43 PD1B756 Humanized PD1B512 PD1H389 PD1L472 PD1B757 Humanized PD1B512 PD1H389 PD1L473

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Table 4

mAb Title VH Title VL SEQ amino acid sequence ID NO: vh VL NA LC PD1B505 PD1H93 PD1L3O 8 14 20 26 PD1B742 PD1H384 PD1L468 9 15 21 27 PD1B743 PD1H384 PD1L469 9 16 21 28 PD1B878 PD1H405 PD1L469 10 16 22 28 PD1B506 PD1H90 PD1L28 44 60 66 82 PD1B75O PD1H388 PD1L470 45 61 67 83 PD1B751 PD1H388 PD1L471 45 62 67 84 PD1B845 PD1H399 PD1L470 46 61 68 83 PD1B846 PD1H400 PD1L470 47 61 69 83 PD1B847 PD1H401 PD1L470 48 61 70 83 PD1B848 PD1H402 PD1L470 49 61 71 83 PD1B849 PD1H403 PD1L470 50 61 72 83 PD1B85O PD1H404 PD1L470 51 61 73 83 PD1B512 PD1H81 PD1L43 94 98 104 108 PD1B756 PD1H389 PD1L472 95 99 105 109 PD1B757 PD1H389 PD1L473 95 100 105 BY

Table 5

mAb Title VH Title VL SEQ polynucleotide sequence ID NO: vh VL HC LC PD1B505 PD1H93 PD1L30 eleven 17 23 29 PD1B742 PD1H384 PD1L468 12 18 24 thirty PD1B743 PD1H384 PD1L469 12 19 24 31 PD1B878 PD1H405 PD1L469 13 19 25 31 PD1B506 PD1H90 PD1L28 52 63 74 85 PD1B750 PD1H388 PD1L470 53 64 75 86 PD1B751 PD1H388 PD1L471 53 65 75 87 PD1B845 PD1H399 PD1L470 54 64 76 86 PD1B846 PD1H400 PD1L470 55 64 77 86 PD1B847 PD1H401 PD1L470 56 64 78 86 PD1B848 PD1H402 PD1L470 57 64 79 86 PD1B849 PD1H403 PD1L470 58 64 80 86 PD1B850 PD1H404 PD1L470 59 64 81 86 PD1B512 PD1H81 PD1L43 96 101 106 111 PD1B756 PD1H389 PD1L472 97 102 107 112 PD1B757 PD1H389 PD1L473 97 103 107 113

Table 6

Antibody HCDR1 HCDR2 HDDR3 LCDR1 LCDR2 LCDR3 PD1B505 2 3 4 5 6 7 PD1B742 2 3 4 5 6 7 PD1B743 2 3 4 5 6 7 PD1B878 2 3 4 5 6 7 PD1B506 32 33 40 41 42 43 PD1B750 32 33 40 41 42 43 PD1B751 32 33 40 41 42 43 PD1B845 32 34 40 41 42 43 PD1B846 32 35 40 41 42 43 PD1B847 32 36 40 41 42 43 PD1B848 32 37 40 41 42 43 PD1B849 32 38 40 41 42 43 PD1B850 32 39 40 41 42 43 PD1B512 88 89 90 91 92 93 PD1B756 88 89 90 91 92 93 PD1B757 88 89 90 91 92 93

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Table 7

Antibody HCDR1 (SEQ ID NO:) HCDR2 (SEQ ID NO:) HDDR3 (SEQ ID NO:) PD1B505 GYTFTDYSMH (SEQ ID NO: 2) WINIETGEPT (SEQ ID NO: 3) DYYGTYFYAMDY (SEQ ID NO: 4) PD1B742 GYTFTDYSMH (SEQ ID NO: 2) WINIETGEPT (SEQ ID NO: 3) DYYGTYFYAMDY (SEQ ID NO: 4) PD1B743 GYTFTDYSMH (SEQ ID NO: 2) WINIETGEPT (SEQ ID NO: 3) DYYGTYFYAMDY (SEQ ID NO: 4) PD1B878 GYTFTDYSMH (SEQ ID NO: 2) WINIETGEPT (SEQ ID NO: 3) DYYGTYFYAMDY (SEQ ID NO: 4) PD1B506 GYTFTTYWMH (SEQ ID NO: 32) EINPNNGGIN (SEQ ID NO: 33) DYYDYGGY (SEQ ID NO: 40) PD1B750 GYTFTTYWMH (SEQ ID NO: 32) EINPNNGGIN (SEQ ID NO: 33) DYYDYGGY (SEQ ID NO: 40) PD1B751 GYTFTTYWMH (SEQ ID NO: 32) EINPNNGGIN (SEQ ID NO: 33) DYYDYGGY (SEQ ID NO: 40) PD1B845 GYTFTTYWMH (SEQ ID NO: 32) EINPNNAGIN (SEQ ID NO: 34) DYYDYGGY (SEQ ID NO: 40) PD1B846 GYTFTTYWMH (SEQ ID NO: 32) EINPNDAGIN (SEQ ID NO: 35) DYYDYGGY (SEQ ID NO: 40) PD1B847 GYTFTTYWMH (SEQ ID NO: 32) EINPNQGGIN (SEQ ID NO: 36) DYYDYGGY (SEQ ID NO: 40) PD1B848 GYTFTTYWMH (SEQ ID NO: 32) EINPNKGGIN (SEQ ID NO: 37) DYYDYGGY (SEQ ID NO: 40) PD1B849 GYTFTTYWMH (SEQ ID NO: 32) EINPNEGGIN (SEQ ID NO: 38) DYYDYGGY (SEQ ID NO: 40) PD1B850 GYTFTTYWMH (SEQ ID NO: 32) EINPNNIGIN (SEQ ID NO: 39) DYYDYGGY (SEQ ID NO: 40) PD1B512 GFSLSTSGMGVS (SEQ ID NO: 88) HIYWDDDKR (SEQ ID NO: 89) KGYYDYGYVMDY (SEQ ID NO: 90) PD1B756 GFSLSTSGMGVS (SEQ ID NO: 88) HIYWDDDKR (SEQ ID NO: 89) KGYYDYGYVMDY (SEQ ID NO: 90) PD1B757 GFSLSTSGMGVS (SEQ ID NO: 88) HIYWDDDKR (SEQ ID NO: 89) KGYYDYGYVMDY (SEQ ID NO: 90)

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Table 8

Antibody LCDR1 (SEQ ID NO:) LCDR2 (SEQ ID NO:) LCDR3 (SEQ ID NO:) PD1B505 TASSSVSSSYLH STSNLAS HQYHRSPLT (SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) PD1B742 TASSSVSSSYLH STSNLAS HQYHRSPLT (SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) PD1B743 TASSSVSSSYLH STSNLAS HQYHRSPLT (SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) PD1B878 TASSSVSSSYLH STSNLAS HQYHRSPLT (SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) PD1B506 KASQNVGTNVA SASYRYS QQYNIYPYT (SEQ ID NO: 41) (SEQ ID NO: 42) (SEQ ID NO: 43) PD1B750 KASQNVGTNVA SASYRYS QQYNIYPYT (SEQ ID NO: 41) (SEQ ID NO: 42) (SEQ ID NO: 43) PD1B751 KASQNVGTNVA SASYRYS QQYNIYPYT (SEQ ID NO: 41) (SEQ ID NO: 42) (SEQ ID NO: 43) PD1B845 KASQNVGTNVA SASYRYS QQYNIYPYT (SEQ ID NO: 41) (SEQ ID NO: 42) (SEQ ID NO: 43) PD1B846 KASQNVGTNVA SASYRYS QQYNIYPYT (SEQ ID NO: 41) (SEQ ID NO: 42) (SEQ ID NO: 43) PD1B847 KASQNVGTNVA SASYRYS QQYNIYPYT (SEQ ID NO: 41) (SEQ ID NO: 42) (SEQ ID NO: 43) PD1B848 KASQNVGTNVA SASYRYS QQYNIYPYT (SEQ ID NO: 41) (SEQ ID NO: 42) (SEQ ID NO: 43) PD1B849 KASQNVGTNVA SASYRYS QQYNIYPYT (SEQ ID NO: 41) (SEQ ID NO: 42) (SEQ ID NO: 43) PD1B850 KASQNVGTNVA SASYRYS QQYNIYPYT (SEQ ID NO: 41) (SEQ ID NO: 42) (SEQ ID NO: 43) PD1B512 RSSKSLLHSNGITYLN QMSNLAS AQNLELPLT (SEQ ID NO: 91) (SEQ ID NO: 92) (SEQ ID NO: 93) PD1B756 RSSKSLLHSNGITYLN QMSNLAS AQNLELPLT (SEQ ID NO: 91) (SEQ ID NO: 92) (SEQ ID NO: 93) PD1B757 RSSKSLLHSNGITYLN QMSNLAS AQNLELPLT (SEQ ID NO: 91) (SEQ ID NO: 92) (SEQ ID NO: 93)

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Table 9

Circuit name VH or VL (SEQ W NO) Amino acid sequence PD1H93 (SEQ ID NO: 8) DVQLQESGPELKKPGETVKISCKASGYTFTDYSMHWVKQAPGKG LKWMGWINIETGEPTYADDFKGRFAFSLETSASTAYLQINNLKNE PD1H384 (SEQ ID NO: 9) QVQLVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRQAPGQ GLEWMGWINIETGEPTYAQGFTGRFVFSLDTSVSTAYLQICSLKAE PD1H405 (SEQ ID NO: 10) QVQLVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRQAPGQ GLEWMGWINIETGEPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAE PD1H90 (SEQ ID NO: 44) QVQLQQPGAELVKPGASVKLSCKASGYTFTTYWMHWVKQRPGQ GLEWIGEINPNNGGINYNEKFKKKATLTVDKSSSTAYMQLSSLTSE PD1H388 (SEQ ID NO: 45) QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWVRQAPG QGLEWMGEINPNNGGINYAQKFQGRVTTLTVDKSISTAYMELSRLR PD1H399 (SEQ ID NO: 46) QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWVRQAPG QGLEWMGEINPNNAGINYAQKFQGRVTTLTVDKSISTAYMELSRLR PD1H400 (SEQ ID NO: 47) QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWVRQAPG QGLEWMGEINPNDAGINYAQKFQGRVTLTVDKSISTAYMELSRLR PD1H401 (SEQ ID NO: 48) QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWVRQAPG QGLEWMGEINPNQGGINYAQKFQGRVTTLTVDKSISTAYMELSRLR PD1H402 (SEQ ID NO: 49) QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWVRQAPG QGLEWMGEINPNKGGINYAQKFQGRVTLTVDKSISTAYMELSRLR PD1H403 (SEQ ID NO: 50) QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWVRQAPG QGLEWMGEINPNEGGINYAQKFQGRVTTLTVDKSISTAYMELSRLR PD1H404 (SEQ ID NO: 51) QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWVRQAPG QGLEWMGEINPNNIGINYAQKFQGRVTLTVDKSISTAYMELSRLR PD1H81 (SEQ ID NO: 94) QVTLKESGPGLLQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKG LEWLAHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITSVDTAD PD1H389 (SEQ ID NO: 95) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKAL EWLAHIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVD PD1L30 (SEQ ID NO: 14) QIVLTQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQQKPGSSPK LWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQY PD1L468 (SEQ ID NO: 15) EIVLTQSPATLSLSPGERATLSCTASSSVSSSYLHWYQQKPGLAPR LLIYSTSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQYH PD1L469 (SEQ ID NO: 16) EIVLTQSPATLSLSPGERATLSCTASSSVSSSYLHWYQQKPGLAPR LLIYSTSNLASGIPDRFSGSGSGTDYTLTISRLEPEDFAVYYCHQYH PD1L28 (SEQ ID NO: 60) DIVMTQSQKFMSTSVRDRVSVTCKASQNVGTNVAWYQQKPGQS PKALIYSASYRYSGVPDRFTGSGSGTDFTLTITNVQSEDLAEYFCQ PD1L470 (SEQ ID NO: 61) DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPEKAP KSLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQY PD1L471 (SEQ ID NO: 62) DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPEKAP KALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQY PD1L43 (SEQ ID NO: 98) DIVMTQAALSNPVTLGTSASISCRSSKSLLHSNGITYLNWYLQKPG QSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYY PD1L472 (SEQ ID NO: 99) DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLNWYLQKPGQ SPQLLIYQMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC PD1L473 (SEQ ID NO: 100) DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLNWYLQKPGQ SPQLLIYQMSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYC

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Table 10

VH chain (polynucleotide SEQ ID NO) Polynucleotide sequence PD1H93 (SEQ ID NO: 11) PD1H384 (SEQ ID NO: 12) PD1H405 (SEQ ID NO: 13) PD1H90 (SEQ ID NO: 52) PD1H388 (SEQ ID NO: 53) GATGTACAGCTTCAGGAGTCAGGACCTGAGCTGAAAGCCTGGA GAGACAGTCAAGATCTCCTGCAAGGCTTTCTGGTTATACCTTCACAG ACTATTCAATGCACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAA AGTGGATGGGCTGGATAAACATTGAGACTGGTGAGCCAACATATG CAGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAA ACCTCTGC CAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACAC GGCTACATATTTCTGTGCTAGAGATTACTACGGTACTTACTTCTAT GCTATGGACTACTGGGGTCAAGGCACCACTCTCACAGTCTCCTCA CAGGTGCAGCTGGTGCAGTCTGGAAGCGAACTGAAGAAACCTGGA GCCTCTGTGAAAGTGTCTTGTAAGGCCAGCGGCTACACCTTCACCG ACTACAGCATGCACTGGGTGCGGCAGGCCCCTGGACAGGGCCTGG AATGGATGGGCTGGATCAACATCGAGACCGGCGAGCCCACCTACG CCCAGGGCTTTACCGGACGGTTCGTGTTCAAGCCTGGATA CATCTGT GTCTACAGCCTATCTGCAGATCTGCTCTCTGAAGGCCGAAGATAC AGCCGTGTACTTCTGCGCCCGGGACTACTACGGCACCTACTTCTAC CCTTTACC GACTACAGCATGCACTGGGTCCGACAGGCTCCAGGACAAGGCTTG GAATGGATGGGCTGGATCAACATCGAGACAGGCGAGCCCACATAC GCCCAGGGCTTTACCGGCAGATTCGTGTTCAGCCTGGACACCTCTG TA CGCCATGGATTACTGGGGCCAGGGCACCCTGGTTACCGTTTCTTCT CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTTGTGAAGCCTGGG GCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCA CCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTG AGTGGATTGGAGAGATTAATCCTAACAATGGTGGTATTAATTACA ATGAGAAGTTCAAGAAGAAGGCCACACTGACTGTA GACAAATCCT CCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACT CTGCGGTCTATTACTGTACAATAGACTATGATTACGGGGGCTA CTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA CAGGTGCAGCTGGTGCAGTCTGGAGCCGAAGTGAAGAAACCTGGA GCCTCTGTGAAAGTGTCTTGTAAGGCCAGCGGCTACACCTTCACCA CCTACTGGATGCACTGGGTGCGGCAGGCCCCTGGACAGGGCCTGG AATGGATGGGCGAGATCAACCCCAACAACGGCGGCATCAACTACG CCCAGAAATTTCAGGGACGGGTGACCCTGACAGTG GATAAGAGCA TCCTACAGCCTACATGGAACTGTCTCGGCTGCGGAGCGATGACA CAGCCGTGTACTGCACCATCGACTACTACGACTACGGCGGCT ACTGGGGCCAGGGAACACTGGTGACAGTGTCTTCT

- 54 043064

PD1H399 (SEQ ID NO: 54) CAGGTGCAGCTGGTGCAGTCTGGAGCCGAAGTGAAGAAACCTGGA GCCTCTGTGAAAGTGTCTTGTAAGGCCAGCGGCTACACCTTCACCA CCTACTGGATGCACTGGGTGCGGCAGGCCCCTGGACAGGGCCTGG AATGGATGGGCGAGATCAACCCCAACAACGCCGGCATCAACTACG CCCAGAAATTTCAGGGACGGGTGACCCTGACAGTGG ATAAGAGCA TCCTACAGCCTACATGGAACTGTCTCGGCTGCGGAGCGATGACA CAGCCGTGTACTGCACCATCGACTACTACGACTACGGCGGCT ACTGGGGCCAGGGAACACTGGTGACAGTGTCTTCT PD1H400 (SEQ ID NO: 55) CAGGTGCAGCTGGTGCAGTCTGGAGCCGAAGTGAAGAAACCTGGA GCCTCTGTGAAAGTGTCTTGTAAGGCCAGCGGCTACACCTTCACCA CCTACTGGATGCACTGGGTGCGGCAGGCCCCTGGACAGGGCCTGG AATGGATGGGCGAGATCAACCCCAACGACGCCGGCATCAACTACG CCCAGAAATTTCAGGGACGGGTGACCCTGACAGTGG ATAAGAGCA TCCTACAGCCTACATGGAACTGTCTCGGCTGCGGAGCGATGACA CAGCCGTGTACTGCACCATCGACTACTACGACTACGGCGGCT ACTGGGGCCAGGGAACACTGGTGACAGTGTCTTCT PD1H401 (SEQ ID NO: 56) CAGGTGCAGCTGGTGCAGTCTGGAGCCGAAGTGAAGAAACCTGGA GCCTCTGTGAAAGTGTCTTGTAAGGCCAGCGGCTACACCTTCACCA CCTACTGGATGCACTGGGTGCGGCAGGCCCCTGGACAGGGCCTGG AATGGATGGGCGAGATCAACCCCAACCAGGGCGGCATCAACTACG CCCAGAAATTTCAGGGACGGGTGACCCTGACAGTG GATAAGAGCA TCCTACAGCCTACATGGAACTGTCTCGGCTGCGGAGCGATGACA CAGCCGTGTACTGCACCATCGACTACTACGACTACGGCGGCT ACTGGGGCCAGGGAACACTGGTGACAGTGTCTTCT PD1H402 (SEQ ID NO: 57) AGGTGCAGCTGGTGCAGTCTGGAGCCGAAGTGAAGAAACCTGGA GCCTCTGTGAAAGTGTCTTGTAAGGCCACGGCTACACCTTCACCA CCTACTGGATGCACTGGGTGCGGCAGGCCCCTGGACAGGGCCTGG AATGGATGGGCGAGATCAACCCCAACAAGGGCGGCATCAACTAC GCCCAGAAATTTCAGGGACGGGTGACCCTGACAGTGG ATAAGAGC ATCCTACAGCCTACATGGAACTGTCTCGGCTGCGGAGCGATGAC ACAGCCGTGTACTACTGCACCATCGACTACTACGACTACGGCGGC

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TACTGGGGCCAGGGAACACTGGTGACAGTGTCTTCT PD1H403 (SEQ ID NO: 58) PD1H404 (SEQ ID NO: 59) CAGGTGCAGCTGGTGCAGTCTGGAGCCGAAGTGAAGAAACCTGGA GCCTCTGTGAAAGTGTCTTGTAAGGCCAGCGGCTACACCTTCACCA CCTACTGGATGCACTGGGTGCGGCAGGCCCCTGGACAGGGCCTGG AATGGATGGGCGAGATCAACCCCAACGAGGGCGGCATCAACTAC GCCCAGAAATTTCAGGGACGGGTGACCCTGACAGTGG ATAAGAGC ATCCTACAGCCTACATGGAACTGTCTCGGCTGCGGAGCGATGAC ACAGCCGTGTACTACTGCACCATCGACTACTACGACTACGGCGGC TACTGGGGCCAGGGAACACTGGTGACAGTGTCTTCT CAGGTGCAGCTGGTGCAGTCTGGAGCCGAAGTGAAGAAACCTGGA GCCTCTGTGAAAGTGTCTTGTAAGGCCAGCGGCTACACCTTCACCA CCTACTGGATGCACTGGGTGCGGCAGGCCCCTGGACAGGGCCTGG AATGGATGGGCGAGATCAACCCCAACAACATCGGCATCAACTACG CCCAGAAATTTCAGGGACGGGTGACCCTGACAGTGG ATAAGAGCA TCCTACAGCCTACATGGAACTGTCTCGGCTGCGGAGCGATGACA CAGCCGTGTACTGCACCATCGACTACTACGACTACGGCGGCT ACTGGGGCCAGGGAACACTGGTGACAGTGTCTTCT CAGGTTACTCTGAAAGAGTCTGGCCCTGGGTTATTGCAGCCCTCCC AGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACT TCTGGTATGGGTGTGAGCTGGATTCGTCAGCCTTTCAGGAAAGGGT PD1H81 CTGGAGTGGCTGGCACACATTTACTGGGATGATGACAAGCGCTAT (SEQ ID NO: 96) AACCCATCCCTGAAGAGCCGGCTCACAATCTCCAAAGATACCTCC AGCAACCAGGTATTCCTCAAGATCACCAGTGTGGACACTGCAGAT ACTGGCACATACTACTGTGTTCGAAAGGGCTACTATGATTACGGCT ATGTAATGGACTACTGGGGTCAAGGGACCACGGTCACCGTCTCCT CA CAGATCACACTGAAAGAATCTGGACCTACACTGGTGAAACCTACA CAGACCCTGACACTGACCTGTACCTTCAGCGGCTTCAGCCTGAGC ACCAGCGGCATGGGCGTGAGCTGGATTCGGCAGCCTCCTGGAAAG PD1H389 GCCCTGGAATGGCTGGCCCACATCTACTGGGACGACGACAAGCGG (SEQ ID NO: 97) TACAGCCCTAGCCTGAAGTCTCGGCTGACAATCACCAAGGATACC TCTAAGAACCAGGTGGTGCTGACAATGACCAACATGGACCCTGTG GACACAGGCACCTACTACTGCGTGCGGAAGGGCTACTACGACTAC GGCTACGTGATGGACTACTGGGGCCAGGGAACACTGGTGACAGTG TCTTCT

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Table 11

VL chain name (polynucleotide SEQ ID NO) cDNA VL PD1L30 (SEQ ID NO: 17) CAAATTGTTTCTCACCCAGTCTCAGCAATCATGTCTGCATCTCTAG GGGAACGGGTCACCATGACCCTGCACTGCCAGCTCAAGTGTAAGTT C AGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCACCAG TATCATCGTTCCCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGC TGAAA PD1L468 (SEQ ID NO: 18) GAGATCGTGCTGACACAAGTCTCCTGCCACACTGTCTCTGTCTCCTG GAGAACGGGCCACACTGAGCTGCACCGCCAGCAGCAGCGTGAGC AGCAGCTACCTGCACTGGTACCAGCAGAAACCTGGACTGGCCCCT CGGCTGCTGATCTACAGCACCAGCAACCTGGCCAGCGGCATCCCT GATCGGTTTTCTGGCAGCGGATCTGGCACAGATTTACACTGACCA TCAGCGGCTGGAACCTGAGGATTTTGCCGTGTACTACTGCCACC AGTACCACCGGAGCCCCCTGACCTTCGGCCAGGGAACAAAGCTGG AAATCAAG PD1L469 (SEQ ID NO: 19) GAGATCGTGCTGACACAAGTCTCCTGCCACACTGTCTCTGTCTCCTG

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GAGAACGGGCCACACTGAGCTGCACCGCCAGCAGCAGCGTGAGC AGCAGCTACCTGCACTGGTACCAGCAGAAACCTGGACTGGCCCCT CGGCTGCTGATCTACAGCACCAGCAACCTGGCCAGCGGCATCCCT GATCGGTTTTCTGGCAGCGGATCTGGCACAGATTACACACTGACC ATCAGCCGGCTGGAACCTGAGGATTTTGCCGTGTACTACTGCCAC CAG TACCACCGGAGCCCCCTGACCTTCGGCCAGGGAACAAAGCTG GAAATCAAG PD1L28 (SEQ ID NO: 63) GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTA AGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGG CACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAA AGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGAT CGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCAT C ACCAATGTGCAGTCTGAAGACTTGGCAGAATATTTCTGTCAGCAA TATAACATCTATCCGTACACGTTCGGATCGGGGACCAAGCTGGAA ATGAAA PD1L470 (SEQ ID NO: 64) GACATCCAGATGACACAGTCTCCTAGCTCTCTGAGCCGCCTCTGTGG GAGATCGGGTGACAATCACCTGCAAGGCCAGCCAGAACGTGGGC ACCAACGTGGCCTGGTACCAGCAGAAACCTGAAAAAGCCCCTAAG AGCCTGATCTACAGCGCCAGCTACCGGTACAGCGGCGTGCCTTCT CGGTTTAGCGGCTCTGGAAGCGGAACAGATTTCACACTGAC CATC TCTAGCCTGCAGCCTGAAGATTTTGCCACATACTACTGCCAGCAGT ACAACATCTACCCCTACACCTTCGGCCAGGGAACAAAGCTGGAAA TCAAG PD1L471 (SEQ ID NO: 65) GACATCCAGATGACACAGTCTCCTAGCTCTCTGAGCCGCCTCTGTGG GAGATCGGGTGACAATCACCTGCAAGGCCAGCCAGAACGTGGGC ACCAACGTGGCCTGGTACCAGCAGAAACCTGAAAAAGCCCCTAAG GCCCTGATCTACAGCGCCAGCTACCGGTACAGCGGCGTGCCTTCT CGGTTTAGCGGCTCTGGAAGCGGAACAGATTTCACACTGAC CATC TCTAGCCTGCAGCCTGAAGATTTTGCCACATACTTTTGCCAGCAGT ACAACATCTACCCCTACACCTTCGGCCAGGGAACAAAGCTGGAAA TCAAG PD1L43 (SEQ ID NO: 101) GATATTGTGATGACTCAGGCTGCACTCTCCAATCCAGTCACTCTTG GAACATCAGCTTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTACA TAGTAATGGCATCACTTATTTGAATTGGTATCTGCAGAAGCCAGG

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CCAGTCTCCTCAGCTCCTGATTTATCAGATGTCCAACCTTGCCTCA GGAGTCCCAGACAGGTTCAGTAGCAGTGGGTCAGGAACTGATTTC ACACTGAGAATCAGCAGAGTGGAGGCTGAGGATGTGGGTGTTTAT TACTGTGCTCAAAATCTAGAACTTCCGCTCACGTTCGGATCGGGG ACCAAGCTGGAAATGAAA PD1L472 (SEQ ID NO: 102) GACATCGTGATGACACAAGTCTCCTCTGTCTCTGCCTGTGACACCTG GCGAACCTGCCTCTATCAGCTGCCGGAGCAGCAAGAGCCTGCTGC ACAGCAACGGCATCACCTACCCTGAACTGGTACCTGCAGAAACCTG GACAGTCTCCTCAGCTGCTGATCTACCAGATGAGCAACCTGGCCA GCGGCGTGCCTGATCGGTTTAGCGGCTCTGGAAGCGGCACAGACT TCAC ACTGAAGATCTCTCGGGTGGAAGCCGAGGACGTGGGAGTGT ACTACTGCGCCCAGAACCTGGAGCTGCCCCTGACCTTCGGAGGCG GAACAAAGGTGGAGATCAAG PD1L473 (SEQ ID NO: 103) GACATCGTGATGACACAAGTCTCCTCTGTCTCTGCCTGTGACACCTG GCGAACCTGCCTCTATCAGCTGCCGGAGCAGCAAGAGCCTGCTGC ACAGCAACGGCATCACCTACCCTGAACTGGTACCTGCAGAAACCTG GACAGTCTCCTCAGCTGCTGATCTACCAGATGAGCAACCTGGCCA GCGGCGTGCCTGATCGGTTTAGCAGCTCTGGAAGCGGCACAGACT TCAC ACTGAAGATCTCTCGGGTGGAAGCCGAGGACGTGGGAGTGT ACTACTGCGCCCAGAACCTGGAGCTGCCCCTGACCTTCGGAGGCG GAACAAAGGTGGAGATCAAG

Table 12

mAb HC HC-PD1B505 (SEQ ID NO: 20) DVQLQESGPELKKPGETVKISCKASGYTFTDYSMHWVKQAPGKGLK WMGWINIETGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATY FCARDYYGTYFYAMDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSVPLYSLSSV VTSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK HCPD1B742, QVQLVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRQAPGQGLE

- 59 043064

PD1B743 (SEQ ID NO: 21) WMGWINIETGEPTYAQGFTGRFVFSLDTSVSTAYLQICSLKAEDTAVY FCARDYYGTYFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK NS PD1B878 (SEQ ID NO: 22) VTVP VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS D IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK NS PD1B506 (SEQ ID NO: 66) QVQLQQPGAELVKPGASVKLSCKASGYTFTTYWMHWVKQRPGQGL EWIGEINPNNGGINYNEKFKKKATLTVDKSSSTAYMQLSSLTSEDSAV YYCTIDYYDYGGYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK NS PD1B750, PD1B751 (SEQ ID NO: 67) QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWVRQAPGQGL EWMGEINPNNGGINYAQKFQGRVTLTVDKSISTAYMELSRLRSDDTA VYYCTIDYYDYGGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV

- 60 043064

EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK NS PD1B845 (SEQ GO NO: 68) VPSS A VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK NS PD1B846 (SEQ ID NO: 69) QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWVRQAPGQGL EWMGEINPNDAGINYAQKFQGRVTLTVDKSISTAYMELSRLRSDDTA VYYCTIDYYDYGGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSS A VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK NS PD1B847 (SEQ GO NO: 70) VP SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK NS PD1B848 QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWVRQAPGQGL

- 61 043064

(SEQ ID NO: 71) EWMGEINPNKGGINYAQKFQGRVTLTVDKSISTAYMELSRLRSDDTA VYYCTIDYYDYGGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKVEPKSCDKTHTCPPCPAPELL GGP PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK NS PD1B849 (SEQ ID NO: 72) QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWVRQAPGQGL EWMGEINPNEGGINYAQKFQGRVTLTVDKSISTAYMELSRLRSDDTA VYYCTIDYYDYGGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSS A VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK NS PD1B850 (SEQ ID NO: 73) PSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK HC PD1B512 (SEQ ID NO: 104) CVRKGYYDYGYVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG NS PD1B756, PD1B757 (SEQ ID NO: 105) VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEW LAHIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTGTYY CVRKGYYDYGYVMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK

- 62 043064

Table 13

mAb LC (SEQ ID NO:) LC-PD1B505 (SEQ GO NO: 26) QIVLTQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQQKPGSSPKLW IYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSP LTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQS GNS QES VTEQDS KDSTYS LS STLTLS KAD YEKH KVYACEVTHQGLSSPVTKSFNRGEC LCPD1B742 (SEQ ID NO: 27) EIVLTQSPATLSLSPGERATLSCTASSSVSSSYLHWYQQKPGLAPRLLI YSTSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQYHRSPLT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACE VTHQGLSSPVTKSFNRGEC LC PD1B743, PD1B878 (SEQ ID NO: 28) EIVLTQSPATLSLSPGERATLSCTASSSVSSSYLHWYQQKPGLAPRLLI YSTSNLASGIPDRFSGSGSGTDYTLTISRLEPEDFAVYYCHQYHRSPLT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACE VTHQGLSSPVTKSFNRGEC LCPD1B506 (SEQ ID NO: 82) PD1B750, PD1B845, PD1B846, PD1B847, PD1B848, PD1B849, LCPD1B850 (SEQ ID NO: 83) LCPD1B751 (SEQ ID NO: 84) LCPD1B512 (SEQ ID NO: 108) LCPD1B756 (SEQ ID NO: 109) DIVMTQSQKFMSTSVRDRVSVTCKASQNVGTNVAWYQQKPGQSPK ALIYSASYRYSGVPDRFTGSGSGTDFTLTITNVQSEDLAEYFCQQYNI YPYTFGSGTKLEMKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE K HKVYACEVTHQGLSSPVTKSFNRGEC DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPEKAPKSL IYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNIYPY EKHKV YACEVTHQGLSSPVTKSFNRGEC DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPEKAPKAL IYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYNIYPYT HKV YACEVTHQGLSSPVTKSFNRGEC DIVMTQAALSNPVTLGTSASISCRSSKSLLHSNGITYLNWYLQKPGQS PQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQN LELPLTFGSGTKLEMKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLNWYLQKPGQSP QLLIYQMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQN LELPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC LCPD1B757 (SEQ ID NO: 110) DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLNWYLQKPGQSP QLLIYQMSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNL ELPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YE KHKVYACEVTHQGLSSPVTKSFNRGEC

- 63 043064

Table 14 HC cDNA (SEQ ID NO:)

HC PD1B505 (SEQ ID NO: 23)

GATGTACAGCTTCAGGAGTCAGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAA

T TCTGTGCTAGATTACTACGGTACTTCTATGCTATGGACTACTGGGGTCAA GGCACCACTCTCACAGTCTCCTCAGCCTCCACCAAGGGCCATCGGTCTTCCCCCTG GCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAA GGACTACTTCCCGAACCGGTGACGGTGTCGTGTGGAACTCAGGCGCCCCTGACCAG CG GCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTCTACTCCCTCAGCAGCG TGGTGACCGTGCCCTCCAGCAGCTTGGGCACCAGACCTACATCTGCAACGTGAATC ACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAA AACTCACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTT CCTCTT CCCCCCAAAACCCAAGGACACCCTCATGATCCCGGACCCCTGAGGTCAC ATGCGTGTGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACG TGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAA CAGCACGTACCGTGTGTGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATG GCAAGGA GTACAAGTGCAAGGTCTCCAACAAAGCCCCTCCCAGCCCCCATCGAGAAA ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCC ATCCCGGGAGGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCT TCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAAC TACAAGACC ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAG CTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGAT GCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTA AA

HC PD1B742, PD1B743 (SEQ ID NO: 24)

Cat CTGTGTCTACAGCCTATCTGCAGATCTGCTCTCTGAAGGCCGAAGATACAGCCG TGTACTTCTGCGCCCGGGACTACTACGGCACCTACTTCTACGCCATGGACTACTGGG GCCAGGGAACACTGGTGACAGTGTCTTCTGCCTCCACCAAGGGCCCATCGGTCTTCC CCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGCACAGCGGCCCTGGGCTGCCTG GTCA AGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGAC CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCTCAG

- 64 043064

CAGCGTGGTGACCGTGCCCTCCAGCAAGCTTGGGCACCCAGACCTACATCTGCAACG TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGT GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTC TCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA GTACAACAGCACGTACCGTGTGTCCTGCACCAGGACTGGCT GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCCTCCCAGCCCCCATCG AGA AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGTGTACACCCTG CCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCCTGACCTGCCTGGTCAA CACCGTGGACAAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCC GTGATGCATGAGGCCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCG GGTAAA

HC PD1B878 (SEQ ID NO: 25)

CAAGTGCAGCTGGTGCAGTCTGGCAGCGAGCTGAAAAAACCTGGCGCCTCCG TGAAGGTGTCCTGCAAGGCTAGCGGCTACACCTTTACCGACTACAGCATGCACTGG GTCCGACAGGCTCCAGGACAAGGCTTGGAATGGATGGGCTGGATCAACATCGAGAC AGGCGAGCCCACATACGCCCAGGGCTTTACCGGCAGATTCGTGTTCAGCCTGGACA CC TCTGTGTCCACCGCCTACCTGCAGATCAGCTCTCTGAAGGCCGAGGATACCGCCG TGTACTTCTGCGCCAGAGACTACTACGGCACCTACTTCTACGCCATGGATTACTGGG GCCAGGGCACCCTGGTTACCGTTTCTTCTGCCTCCACCAAGGGCCCATCGGTCTTCC CCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGCACAGCGGCCCTGGGCTGCCTG GTCAAG GACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGAC CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAG CAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGT GACAAAACTCA CACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTC AGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCCTGA GGTCACCATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT GGTACGTGGACGGCGTGGAGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA GTACAACAGCACGTA CCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCG AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG

- 65 043064

CCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGA ACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACA GCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCT CC GTGATGCATGAGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCG GGTAAA

HC PD1B506 (SEQ ID NO: 74)

CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTTGTGAAGCCTGGGGCTTCAG TGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCACCTACTGGATGCACTGGG A ATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGT CTATTACTGTACAATAGACTATGATTACGGGGGCTACTGGGGCCAAGGCACCA CTCTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCT CCTCCAAGAGCACCTCTGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAG GACTAC TTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCCGCCCTGACCAGCGGCGTGCA CACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGAC CGTGCCCTCCAGCAAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGC CCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACT CAC ACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTC CCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGT GGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACG GCGTGGAGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGC AC GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGG AGTACAAGTGCAAGGTCTCCAACAAAGCCCCTCCCAGCCCCCATCGAGAAAACCATC TCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCG GGAGGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATC CGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATG AGCTCTGCACAACCACTACACGCAGAAGCCTCTCCCTGTCTCCGGGTAAA

HC PD1B750, PD1B751 (SEQ ID NO: 75)

CAGGTGCAGCTGGTGCAGTCTGGAGCCGAAGTGAAGAAACCTGGAGCCTCT GTGAAAGTGTCTTGTAAGGCCAGCGGCTACACCTTCACCACCTACTGGATGCACTGG GTGCGGCAGGCCCCTGGACAGGGCCTGGAATGGATGGGCGAGATCAACCCCAACA

- 66 043064

ACGGCGGCATCAACTACGCCCAGAAATTTCAGGGACGGGTGACCCTGACAGTGGAT AAGAGCATCTCTACAGCCTACATGGAACTGTCTCGGCTGCGGAGCGATGACACAGC CCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC TACTTCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCCTGACCAGCGGCGT GCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT CA AGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACT CACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGACCGTCAGTCTTCCTC TTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGA CGGCGTGGAGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGC ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAA GGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTA TCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTC ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA TG AGGCTCTCGCACAACCACTACACCGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

HC PD1B845 (SEQ ID NO: 76)

CAGGTGCAGCTGGTGCAGTCTGGAGCCGAAGTGAAGAAACCTGGAGCCTCT GTGAAAGTGTCTTGTAAGGCCAGCGGCTACACCTTCACCACCTACTGGATGCACTGG GTGCGGCAGGCCCCTGGACAGGGCCTGGAATGGATGGGCGAGATCAACCCCAACA ACGCCGGCATCAACTACGCCCAGAAATTTCAGGGACGGGTGACCCTGACAGTGGAT AAG AGCATCTCTACAGCCTACATGGAACTGTCTCGGCTGCGGAGCGATGACACAGC CGTGTACTACTGCACCATCGACTACTACGACTACGGCGGCTACTGGGGCAGGGAA CACTGGTGACAGTGTCTTCTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCAC CCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC TACT TCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCCGCCCTGACCAGCGGCGT GCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTC

- 67 043064

TTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGA CGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGC ACGTACCGTGTGTGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGC AA GGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCA TCTCCAAAGCCAAAGGGCAGCCCCGAGAACCAACAGGTGTACACCCTGCCCCCATCC AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTC ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

HC PD1B846 (SEQ ID NO: 77)

CAGGTGCAGCTGGTGCAGTCTGGAGCCGAAGTGAAGAAACCTGGAGCCTCT GTGAAAGTGTCTTGTAAGGCCAGCGGCTACACCTTCACCACCTACTGGATGCACTGG GTGCGGCAGGCCCCTGGACAGGGCCTGGAATGGATGGGCGAGATCAACCCCAACG ACGCCGGCATCAACTACGCCCAGAAATTTCAGGGACGGGTGACCCTGACAGTGGAT AAG AGCATCTCTACAGCCTACATGGAACTGTCTCGGCTGCGGAGCGATGACACAGC CGTGTACTACTGCACCATCGACTACTACGACTACGGCGGCTACTGGGGCAGGGAA CACTGGTGACAGTGTCTTCTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCAC CCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC TACT TCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCCGCCCTGACCAGCGGCGT GCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTC TTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGA CGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGC ACGTACC GTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAA GGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCA TCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCC CGGGAGGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTA TCCCAGC GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTC ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA

- 68 043064

TGAGGCCTCTGCACAACCACTACACCGCAGAAGCCTCTCCCTGTCTCCGGGTAAA HC PD1B847 (SEQ ID NO: 78) CAGGTGCAGCTGGTGCAGTCTGGAGCCGAAGTGAAGAAACCTGGAGCCTCT

GTGAAAGTGTCTTGTAAGGCCAGCGGCTACACCTTCACCACCTACTGGATGCACTGG GTGCGGCAGGCCCCTGGACAGGGCCTGGAATGGATGGGCGAGATCAACCCCAACCA GGGCGGCATCAACTACGCCCAGAAATTTCAGGGACGGGTGACCCTGACAGTGGATA AGAGCATCTCTACAGCCTACATGGAACTGTCTCGGCTGCGGAGCGATGACACAG CC GTGTACTACTGCACCATCGACTACTACGACTACGGCGGCTACTGGGGCCAGGGAAC ACTGTGACAGTGTCTTCTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACC CTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACT ACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCCTGACCAGCG GCGTG CACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTCTACTCCCTCAGCAGCGTGTGGTG ACCGTGCCCTCCAGCAAGCTTGGGCACCAGACCTACATCTGCAACGTGAATCACAA G GAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCA TCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAAGGTGTACACCCTGCCCCCATCC GACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTC ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

HC PD1B848 (SEQ ID NO: 79)

CAGGTGCAGCTGGTGCAGTCTGGAGCCGAAGTGAAGAAACCTGGAGCCTCT GTGAAAGTGTCTTGTAAGGCCAGCGGCTACACCTTCACCACCTACTGGATGCACTGG GTGCGGCAGGCCCCTGGACAGGGCCTGGAATGGATGGGCGAGATCAACCCCAACA AGGGCGGCATCAACTACGCCCAGAAATTTCAGGGACGGGTGACCCTGACAGTGGAT AAGAGCATCTCTACAGCCTACATGGAACTGTCTCGGCTGCGGAGCGATGACACAGC CGTGTACTACTGCACCATCGACTACTACGACTACGGCGGCTACTGGGGCCAGGGAA CACTGGTGACAGTGTCTTCTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCAC CCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC

- 69 043064

TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCCTGACCAGCGGCGT GCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGTGT A T CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTC ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCACTACACGCAGAAGCCTCTCCCTGTCTCCGGGTAAA

PD1B849 (SEQ ID NO: 80)

CAGGTGCAGCTGGTGCAGTCTGGAGCCGAAGTGAAGAAACCTGGAGCCTCT GTGAAAGTGTCTTGTAAGGCCAGCGGCTACACCTTCACCACCTACTGGATGCACTGG GTGCGGCAGGCCCCTGGACAGGGCCTGGAATGGATGGGCGAGATCAACCCCAACG AGGGCGGCATCAACTACGCCCAGAAATTTCAGGGACGGGTGACCCTGACAGTGGAT AAGAGCATCTCTACAGCCTACATGGAACTGTCTCGGCTGCGGAGCGATGACACAGC CGTGTACTACTGCACCATCGACTACTACGACTACGGCGGCTACTGGGGCAGGGAA CACTGGTGACAGTGTCTTCTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCAC CCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCCTGACCAGCGGCGT GCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACA AGCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACT CA CACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTC TTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGA CGGCGTGGAGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGC ACG TACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAA GGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCA

- 70 043064

TCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCC CGGGAGGATGACCAAGAACCAGGTCAGCCCTGACCTGCCTGGTCAAAGGCTTCTA TCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTACAGCAAGCTC ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

HC PD1B850 (SEQ ID NO: 81)

CAGGTGCAGCTGGTGCAGTCTGGAGCCGAAGTGAAGAAACCTGGAGCCTCT GTGAAAGTGTCTTGTAAGGCCAGCGGCTACACCTTCACCACCTACTGGATGCACTGG GTGCGGCAGGCCCCTGGACAGGGCCTGGAATGGATGGGCGAGATCAACCCCAACA ACATCGGCATCAACTACGCCCAGAAATTTCAGGGACGGGTGACCCTGACAGTGGAT AAG AGCATCTCTACAGCCTACATGGAACTGTCTCGGCTGCGGAGCGATGACACAGC CGTGTACTACTGCACCATCGACTACTACGACTACGGCGGCTACTGGGGCAGGGAA CACTGGTGACAGTGTCTTCTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCAC CCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC TACT TCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCCGCCCTGACCAGCGGCGT GCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTC TTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGA CGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGC ACGTACC GTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAA GGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCA TCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCC CGGGAGGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTA TCCCAGC GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTC ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCACTACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

HC PD1B512 (SEQ ID NO: 106)

CAGGTTACTCTGAAAAGTCTGGCCCTGGGTTATTGCAGCCCTCCCAGACCC TCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTGTGAG CTGGATTCGTCAGCCTTTCAGGAAAGGGTCTGGAGTGGCTGGCACACATTTACTGGG

- 71 043064

ATGATGACAAGCGCTATAACCCATCCCTGAAGCCGGCTCACAATCTCCAAAGAT ACCTCCAGCAACCAGGTATTCCTCAAGATCACCAGTGTGGACACTGCAGATACTGG CCCCCTGGCACCCCTCCCAAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCT GGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGA CAAC GTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTG TGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGT CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTG AGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAACAAAGCCGCGGGAGGAGC AGTACAACAGCACGTACCGTGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGG TGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGT CAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCT ACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGC TCCGTGATGCATGAGGCCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCT CCGGGTAAA

HC PD1B756, PD1B757 HC (SEQ ID NO: 107)

CAGATCACACTGAAAGAATCTGGACCTACACTGGTGAAACCTACACAGACCC TGACACTGACCTGTACCTTCAGCGGCTTCAGCCTGAGCACCAGCGGCATGGGCGTG AGCTGGATTCGGCAGCCTCCTGGAAAGGCCCTGGAATGGCTGGCCCACATCTACTG GGACGACGACAAGCGGTACAGCCCTAGCCTGAAGTCTCGGCTGACAATCACCAAGG ATACC TCTAAGAACCAGGTGGTGCTGACAATGACCAACATGGACCCTGTGGACACA GGCACCTACTACTGCGTGCGGAAGGGCTACTACGACTACGGCTACGTGATGGACTA CTGGGGCAGGGAACACTGGTGACAGTGTCTTCTGCCTCCACCAAGGGCCCATCGG TCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT GCC TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCC CTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCC TGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGA CCGTCAGTCTTCCTCTTCCCCCAAAAACCCAAGGACACCCTCATGATCTCCCGGACC CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAG GAGCAG TACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGA CTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCC CCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTAC ACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCT GGTCAAAGGCTT CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGC CGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCC TCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCA TGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTG TCTCCGGGTA AA

- 72 043064

Table 15 LC cDNA (SEQ ID NO:)

LC PD1B505 (SEQ ID NO: 29)

CAAATTGTTTCTCACCCAGTCTCAGCAATCATGTCTGCATCTCTAGGGGAACG GGTCACCATGACCTGCACTGCCAGCTCAAGTGTAAGTTCCAGTTACTTGCACTGGTA CCAGCAGAAGCCAGGATCCTCCCCAAACTCTGGATTTATAGCACATCCAACCTGG CTTCTGGAGTCCCAGCTCGCTTCAGTGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCA CA ATCAGCAGCATGGAGGCCTGAAGATGCTGCCACTTATTACTGCCACCAGTATCATC GTTCCCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGTACGGTGGCT GCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCC GCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGCAGGACAG CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACG AGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTC ACAAAAGAGCTTCAACAGGGGAGAGTGT

LC PD1B742 (SEQ ID NO: 30)

GAGATCGTGCTGACACAAGTCTCCTGCCACACTGTCTCTGTCTCCTGGAGAAC GGGCCACACTGAGCTGCACCGCCAGCAGCAGCGTGAGCAGCAGCTACCTGCACTGG TACCAGCAGAAACCTGGACTGGCCCCTCGGCTGCTGATCTACAGCACCAGCAACCT GGCCAGCGGCATCCCTGATCGGTTTTCTGGCAGCGGATCTGGCACAGATTTTACACT GACCAT CAGCCGGCTGGAACCTGAGGATTTTGCCGTGTACTACTGCCACCAGTACCA CCGGAGCCCCCTGACCTTCGGCCAGGGAACAAAGCTGGAAATCAAGCGTACGGTGG

- 73 043064

CTGCACCATCTGTCTTCATCTTCCCGCCATCTGAGCAGTTGAAATCTGGAACTG CCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGA AGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGAC AGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTA CG AGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCG TCACAAAAGGCTTCAACAGGGGAGAGTGT

LC PD1B743, PD1B878 (SEQ ID NO: 31)

GAGATCGTGCTGACACAAGTCTCCCGCCACACTGTCACTGTCTCCAGGCGAAA GAGCCACACTGAGCTGTACCGCCAGCAGCTCTGTGTCCAGCAGCTACCTGCACTGGT ATCAGCAGAAGCCTGGACTGGCCCCTCGGCTGCTGATCTACAGCACAAGCAATCTG GCCAGCGGCATCCCCGATAGATTTTCCGGCTCTGGAAGCGGCACCGACTACACCCCT GACAATCAGC AGACTGGAACCCGAGGACTTCGCCGTGTACTACTGCCACCAGTACC ACAGAAGCCCTCTGACCTTTGGCCAGGGCACCAAGCTGGAAATCAAGCGTACGGTG GCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACT GCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGG AAGGTGGATAACGCCCTCCA ATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGA CAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACT ACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCC GTCACAAAGAGCTTCAACAGGGGAGAGTGT

LC PD1B506 (SEQ ID NO: 85)

GACATTGTGATGACCAGTCTCAAAAATTCATGTCCACATCAGTAAGAGACA GGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGCACTAATGTAGCCTGGTAT CAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTA CAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCAC CAT CACCAATGTGCAGTCTGAAGACTTGGCAGAATATTTCTGTCAGCAATATAACAT CTATCCGTACACGTTCGGATCGGGGACCAAGCTGGAAATGAAACGTACGGTGGCTG CAATCGGGTAACTCCCAGGAGAGTGTCACAGCAGGACAG CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACG AGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTC ACAAAAGAGCTTCAACAGGGGAGAGTGT

LC PD1B750, PD1B845, PD1B846, PD1B847, PD1B848, PD1B849, PD1B850 (SEQ ID NO: 86)

GACATCCAGATGACACAGTCTCCTAGCTCTCTGAGCCGCCTCTGTGGGAGATC

- 74 043064

GGGTGACAATCACCTGCAAGGCCAGCCAGAACGTGGGCACCAACGTGGCCTGGTAC CAGCAGAAACCTGAAAAAGCCCCTAAGAGCCTGATCTACAGCGCCAGCTACCGGTA CAGCGGCGTGCCTTCTCGGTTTAGCGGCTCTGGAAGCGGAACAGATTTCACACTGAC CATCTCTAGCCTGCAGCCTGAAGATTTTGCCACATACTACTGCCAGCAGTACAACAT C TACCCCTACACCTTCGGCCAGGGAACAAAGCTGGAAATCAAGCGTACGGTGGCTG CACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCT CTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAG GTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG CAAGGACA GCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACG AGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTC ACAAAGAGCTTCAACAGGGGAGAGTGT

LC PD1B751 (SEQ ID NO: 87)

GACATCCAGATGACACAGTCTCCTAGCTCTCTGAGCCGCCTCTGTGGGAGATC GGGTGACAATCACCTGCAAGGCCAGCCAGAACGTGGGCACCAACGTGGCCTGGTAC CAGCAGAAACCTGAAAAAGCCCCTAAGGCCCTGATCTACAGCGCCAGCTACCGGTA CAGCGGCGTGCCTTCTCGGTTTAGCGGCTCTGGAAGCGGAACAGATTTCACACTGAC CATCTCTAGCCTGCAGCCTGAAGATTTTGCCACATACTTTTGCCAGCAGTACAACAT CTACCCCTACACCTTCGGCCAGGGAACAAAGCTGGAAATCAAGCGTACGGTGGCTG TCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACG AGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTC ACAAAAGAGCTTCAACAGGGGAGAGTGT

LCPD1B512(SEQIDNO: 111)

GATATTGTGATGACTCAGGCCTGCACTCTCCAATCCAGTCACTCTTGGAACATC AGCTTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTACATAGTAATGGCATCACTTA TTTGAATTGGTATCTGCAGAAGCCAGGCCAGTCTCCTCAGCTCCTGATTTATCAGAT GTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTAGCAGTGGGTCAGGAACTG ATT TCACACTGAGAATCAGCAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTG CTCAAAATCTAGAACTTCCGCTCACGTTCGGATCGGGGACCAAGCTGGAAATGAAA CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAA GTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCAC AGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC

- 75 043064

AAAGCAGACTACGAGAAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCT GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

LC PD1B756 (SEQ ID NO: 112)

ACCTGAACTGGTACCTGCAGAAACCTGGACAGTCTCTCAGCTGCTGATCTACCAGA TGAGCAACCTGGCCAGCGGCGTGCCTGATCGGTTTAGCGGCTCTGGAAGCGGCACA GACT TCACACTGAAGATCTCTCGGGTGGAAGCCGAGGACGTGGGAGTGTACTACTG CGCCCAGAACCTGGAGCTGCCCCTGACCTTCGGAGGCGGAACAAAGGTGGAGATCA AGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGA AATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGGCCA AAGTACAG TGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTC ACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAAGCACCCTGACGCTGA GCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGC CTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

LC PD1B757 (SEQ ID NO: 113)

ACCTGAACTGGTACCTGCAGAAACCTGGACAGTCTCTCAGCTGCTGATCTACCAGA TGAGCAACCTGGCCAGCGGCGTGCCTGATCGGTTTAGCAGCTCTGGAAGCGGCACA GACT TCACACTGAAGATCTCTCGGGTGGAAGCCGAGGACGTGGGAGTGTACTACTG CGCCCAGAACCTGGAGCTGCCCCTGACCTTCGGAGGCGGAACAAAGGTGGAGATCA AGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGA AATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGGCCA AAGTACAG TGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTC ACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAAGCACCCTGACGCTGA GCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGC CTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

The cDNA sequences encoding the VH, VL, HC, and LC regions of an antibody provided herein can be codon-optimized for expression in a variety of cells, such as for expression in HEK or CHO cells. Additional cDNA sequences that can be used to encode and express the VH, VL, HC, and LC regions of antibodies PD1B878 and PD1B849 are shown below.

SEQ ID NO: 132 cDNA VH PD1B878

CAAGTGCAGCTGGTGCAGTCTGGCAGCGAGCTGAAAAAACCTGGCGCCTCCG

- 76 043064

TGAAGGTGTCCTGCAAGGCCTAGCGGCTACACCTTTACCGACTACAGCATGCACTGGG TCCGACAGGCTCCAGGACAAGGCTTGGAATGGATGGGCTGGATCAACATCGAGACA GGCGAGCCCACATACGCCCAGGGCTTTACCGGCAGATTCGTGTTCAGCCTGGACACC TCTGTGTCCACCGCCTACCTGCAGATCAGCTCTCTGAAGGCCGAGGATACCGCCG TG TACTTCTGCGCCAGAGACTACTACGGCACCTACTTCTACGCCATGGATTACTGGGGC CAGGGCACCCTGGTTACCGTTTCTTCT

SEQ ID NO: 133 cDNA VL PD1B878

GAGATCGTGCTGACACAAGTCTCCCGCCACACTGTCACTGTCTCCAGGCGAAA GAGCCACACTGAGCTGTACCGCCAGCAGCTCTGTGTCCAGCAGCTACCTGCACTGGT ATCAGCAGAAGCCTGGACTGGCCCCTCGGCTGCTGATCTACAGCACAAGCAATCTG GCCAGCGGCATCCCCGATAGATTTTCCGGCTCTGGAAGCGGCACCGACTACACCCTG ACAATCAGCAG ACTGGAACCCGAGGACTTCGCCGTGTACTACTGCCACCAGTACCA CAGAAGCCCTCTGACCTTTGGCCAGGGCACCAAGCTGGAAATCAAG

SEQ ID NO: 134 cDNA HC PD1B878

CAAGTGCAGCTGGTGCAGTCTGGCAGCGAGCTGAAAAAACCTGGCGCCTCCG TGAAGGTGTCCTGCAAGGCTAGCGGCTACACCTTTACCGACTACAGCATGCACTGGG TCTGTGTCCACCGCCTACCTGCAGATCAGCTCTCTGAAGGCCGAGGATACCGCCGTG TACTTCTGCGCCAGAGACTACTACGGCACCTACTTCTACGCCATGGATTACTGGGGC CAGGGCACCCTGGTTACCGTTTCTTCTGCCTCCACCAAGGCCCATCGGTCTTCCCCC TGGCACCTCCTCCAAGAGCACCTCTGGGGCACAGCGGCCCTGGGCTGCCTGGTC A ACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTT CCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCAC ATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACG TGGACGGCGTGGAGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAA CAG CACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGG CAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCCTCCCAGCCCCCATCGAGAAAA ATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGC

SEQ ID NO: 135 cDNA LC PD1B878

GAGATCGTGCTGACACAAGTCTCCCGCCACACTGTCACTGTCTCCAGGCGAAA

- 77 043064

GAGCCACACTGAGCTGTACCGCCAGCAGCTCTGTGTCCAGCAGCTACCTGCACTGGT ATCAGCAGAAGCCTGGACTGGCCCCTCGGCTGCTGATCTACAGCACAAGCAATCTG GCCAGCGGCATCCCCGATAGATTTTCCGGCTCTGGAAGCGGCACCGACTACACCCTG ACAATCAGCAGACTGGAACCCGAGGACTTCGCCGTGTACTACTGCCACCAGTACCA CAGAAGCC CTCTGACCTTTGGCCAGGGCACCAAGCTGGAAATCAAGCGTACGGTGG CTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTG CAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTA CGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCG TCACAAAAGGCTTCAACAGGGGAGAGTGT

SEQ ID NO: 136 cDNA VH PD1B849

CAAGTGCAGCTGGTGCAATCTGGCGCCGAAGTGAAAAAGCCTGGCGCCTCTG TGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTTACCACCTACTGGATGCACTGGG GAGCATCAGCACCGCCTACATGGAACTGAGCCGGCTGAGATCCGATGACACCGCCG TGTACTACTGCACCATCGACTACTACGACTACGGCGGCTATTGGGGCCAGGGCACAC TGGTTACAGTGTCCTCT

SEQ ID NO: 137 cDNA VL PD1B849

GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCTCTGTGGGCGATA GAGTGACCATCACATGCAAGGCCAGCCAGAACGTGGGCACCAATGTGGCCTGGTAT CTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACA TCTACCCCTACACCTTCGGCCAGGGCACCAAGCTGGAAATCAAG

SEQ ID NO: 138 cDNA HC PD1B849

CAAGTGCAGCTGGTGCAATCTGGCGCCGAAGTGAAAAAGCCTGGCGCCTCTG TGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTTACCACCTACTGGATGCACTGGG T CCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCCTGACCAGCGGCGTGCAC ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACC GTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCC AGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCAACAC ATGC CCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTTCCTCTTCCC

- 78 043064

CCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCG TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTA CCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGA GT ACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCA GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGAC CAGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGT GGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGG CTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

SEQ ID NO: 139 cDNA LC PD1B849

GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCTCTGTGGGCGATA GAGTGACCATCACATGCAAGGCCAGCCAGAACGTGGGCACCAATGTGGCCTGGTAT CTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACA TCTACCCCTACACCTTCGGCCAGGGCACCAAGCTGGAAATCAAGCGTACGGTGGCT GCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCC ATCGGGTAACTCCCAGGAGAGTGTCACAGCAGGACAG CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACG AGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTC ACAAAGAGCTTCAACAGGGGAGAGTGT

In FIG. 2A and 2B show the amino acid sequence alignment of the VH and VL mAb line PD1B505, respectively.

In FIG. 3A and 3B show the amino acid sequence alignment of the VL and VL mAb line PD1B506, respectively.

In FIG. 4A and 4B show the amino acid sequence alignment of the VH and VL mAb line PD1B512, respectively.

Example 4 Humanized anti-PD-1 antibodies inhibit antigen-specific T cells.

Selected humanized antibodies were characterized by their ability to inhibit activated T cells in a secondary CMV-specific response assay (CMV-PBMC assay described in Example 1). The parent antibodies PD1B505 and PD1B506 demonstrated robust inhibition of activated T cells when assessed on three different donors in several separate experiments, as shown in Table 1. 16, in which the results are presented as percentage (%) inhibition of T cell proliferation at a mAb concentration of 10 μg/ml. In FIG. 5A shows the mean percent inhibition and standard deviation for PD1B505 (57.8%±9.5%) and PD1B506 (77.0%±7.8%) compared to the isotype control (human IgG1) (-9.5%± 28.8%. Humanized antibodies PD1B743, PD1B750 and PD1B756 also inhibited T-cell activation, as shown in table. 17 as percent inhibition at 10 µg/mL. In FIG. 5B shows the percentage inhibition and standard deviation for PD1B743 (58.0%±11.3%), PD1B750 (65.9%±13.2%), PD1B756 (36.7%±15.5%), compared with isotype control (human IgG1) (3.5%±25.6%). Results above 25% standard deviation were excluded from the analysis. The engineered PD1B878 and PD1B849 antibodies similarly inhibited activated T cells, as shown in Table 1. 18. In FIG. 5C shows mean percent inhibition and standard deviation for PD1B878 (78.3%±18.1%) and PD1B849 (69.0%±4.0%) compared to isotype control (human IgG1) (4.1%±28 .7%).

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Table 16

PD1B505 PD1B506 Isotype Standard Donor Average Standard off Average Standard off Average off D1 86.5 9.0 95.2 1.4 4.7 13.3 D2 74.8 0.8 100.1 3.3 13.2 12.9 D1 77 23.0 98 9.8 4.2 33.6 D2 87.7 3.7 100.0 5.8 -16.6 30.6 D3 0.7 8.3 49.5 11.4 6.1 5.7 D1 77.1 4.1 87.8 4.0 -33.1 59.1 D1 69.7 5.4 85.2 2.7 3.1 24.5 D3 63.4 4.9 101.4 8.7 -23.5 44.3 D1 84.5 7.2 86.4 6.5 -35.6 60.4 D1 76.6 2.5 103.1 5.2 -17.7 28.1 D1 49.8 12.7 64.2 11.5 -14.9 19.5 D1 -7.0 17.1 3.8 12.1 -20.0 29.3 D2 49.1 16.6 76.6 6.8 -0.9 28.1 D1 20.1 17.0 26.2 20.7 -2.0 14.2

Table 17

PD1B743 PD1B750 PD1B756 Isotype Standard Standard Standard Standard Donor Average off Average off Average off Average off D1 90.1 3.1 97.2 10.0 59.3 18.2 -17.7 28.1 D3 21.1 13.8 74.9 16.7 27.4 14.2 -6.6 17.7 D4 30.5 23.6 24.2 15.5 0.3 17.1 2.7 7.7 D5 64.0 5.9 75.0 9.9 69.3 12.8 14.1 35.6 D1 66.7 14.8 58.1 19.8 27.4 15.4 -13.1 45.2 D3 35.1 8.2 55.6 5.8 0.5 20.8 D1 84.2 2.2 72.1 18.8 43.1 17.5 D1 58.4 8.4 64.7 7.3 5.8 31.6 D1 72.1 21.4 71.6 14.8 2.5 25.9

Table 18

PD1B878 PD1B849 Isotype Donor Average Standard off Average Standard off Average Standard off D1 64.3 2.5 5.8 31.6 D1 78.3 18.1 73.7 5.6 2.5 25.9

Example 5 Anti-PD-1 antibodies bind to human PD-1 with high affinity.

The affinity of the original and humanized antibodies to PD-1 was measured using SPR, as described in example 1.

In table. 19 shows the results of affinity measurements. Antibodies bound PD-1 with a KD value ranging from 5.2 x 10 ^-8 M and 2.1 x 10 ^-8 M.

Table 19

mAh Ka (1/Ms) Kd (1/s) K _D (M) PD1B505 8.11E+04 2.36E - 03 2.91E-08 PD1B742 PD1B743 9D5E+04 3,OOE-O3 3.28E - 08 PD1B878 5.98E+04 3.51E-O3 5.89E - 08 PD1B506 1.95E+05 1.02E-02 5.24E - 08 PD1B750 PD1B751 3.59E+05 8.32E - 03 2.32E - 08 PD1B845* 3.51E+05 8.43Е-03 2.40E - 08 PD1B846* 3.49E+05 7.53Е-03 2D6E-08 PD1B847* 3.03E+05 7.44E - 03 2.46E - 08 PD1B848* 2.53E+05 9.91Е-03 3.92E - 08 PD1B849 1.88E+05 8.83E-O3 4.71Е-08 PD1B85O* ZDZE+05 7D9E-03 2,ZOE - 08 PD1B512 PD1B756 1D8E+05 2D4E-03 1.81E - 08 PD1B757 * Binding evaluated for crude supernatants

- 80 043064

Example 6 Anti-PD-1 antibodies block the PD-1/ligand interaction in different ways.

Ligand blocking was analyzed by evaluating the effect of antibodies on clustering of cells overexpressing either PD-1 or PD-1 ligand (PD-L1 or PD-L2) using the protocol described in Example 1. Lower percentage (%) of doubles detected positive events indicated that the mAb under study blocked PD-1 binding to the ligand under study.

In FIG. 6A shows the percentage of PD-1-HEK and PD-L1-HEK cell clusters remaining after cell treatment with PD1B743, PD1B750, or PD1B756 antibodies. PD1B743 and PD1B756 did not block PD-L1 binding to PD-1, while PD1B750 blocked binding. Similarly, PD1B743 and PD1B756 did not block PD-L2 binding to PD-1, while PD1B750 blocked binding (FIG. 6B). In the experiments, a known ligand-blocking mAb to PD-1 was used as a positive control.

Example 7 Grouping of Antibodies by Epitopes.

In the original matrix analyzes using chimeric antibodies and the protocol described in example 1, identified five different epitope groups. Groups 4 and 5 did not cross-compete with any other groups. Groups 1, 2 and 3 were partially overlapping; group 1 competed with group 2 and 3, and groups 2 and 3 competed with group 1. Groups 1, 2, 3, and 4 did not block PD-L1 binding to PD-1, while group 5 blocked PD-L1/PD- 1. In FIG. 7 shows the individual epitope groups and the antibodies of each group.

A control experiment was performed with the addition of an isotype control as the first antibody to demonstrate how the second antibody would itself bind to human PD-1. Then, in the competition experiment, the association signal (nm) of the second antibody was obtained 180 seconds after the start of this step. The signal of each potentially competing antibody was compared to the mean of the signal from 3 runs of the same antibody added after the isotype control. The ratio of these signals was determined. If the ratio was below 0.7, then it was considered that the second antibody could not bind to human PD-1, and that the two antibodies share the same epitope. If the ratio was greater than 0.7, then it was considered that the second antibody could bind in the presence of the first antibody, and therefore the two antibodies bind to non-competing epitopes.

Humanization and engineering of post-translational modifications (PTMs) is not expected to result in epitope shift, and therefore PD1B743, PD1B742, and PD1B878 are expected to bind to the same epitope as the original group 1 PD1B505 chimeric antibody. Similarly, it is expected that PD1B750, PD1B751, and PD1B849 will bind to the same epitope as the parent group 5 PD1B506 chimeric antibody, and PD1B756 is also expected to bind to the same epitope as the parent PD1B512 chimeric group 2 antibody.

The PD1B503 antibody contains the VH and VL sequences of SEQ ID NOs: 114 and 115, respectively.

The PD1B517 antibody contains the VH and VL sequences of SEQ ID NOs: 116 and 117, respectively.

SEQ ID NO: 114 VH PD1B503 (PD1H96)

QVQLQQSGELVKPGASVKLSCKASGYTFTSYDINWVRQRPEQGLEWIGWIFPG DGSTKYNEKFKGKATLTTDKSSSTAYMQFSRLTSEDSAVYFCARGGMRQLGRFVYWG QGTTLTVSS

SEQ ID NO: 115 VL PD1B503 (PD1L32)

DIVLTQSPS S LS AS LGERVSLTCRAS QEIS GYLS WLQQKPDGTIKRLIY AASTLDS G

VPKRFSGSRSGSDYSLTISSLESEDFADYYCLQYASNPYTFGGGTKLEIK

SEQ ID NO: 116 VH PD1B517 (PD1H73)

EVQLQQSGAELVKPGASVKLSCTASGFNVKDTYFHWVKQRPDQGLEWIGRIVS angdtkyapklqdkatittdtssntaylqlsrltsedtavyycvliyygfeegdfwgq GTTLTVSS

SEQ ID NO: 117 VL PD1B517 (PD1L34)

DIVMTQSPSSLSASLGDTITITCHASQNINVWLSWYQQKPGNVPKLLIYKASNLH

TGVPSRFSGSGSGTGFTLNISSLQPEDIATYYCQQGQSFPLTFGAGTKLELK

Example 8 Affinity Maturation of PD1B878.

To increase the binding affinity of PD1B878, CDR scans were performed for both heavy and light chains. Non-combinatorial libraries have been developed to diversify each of the positions of all six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3).

Briefly, Fab libraries were constructed in a pIX phage Fab display system as described in WO 2009/085462, Shi et al., J. Mol. Biol. 397: 385-396 (2010) and Tornetta et al. J. Immunol. Methods 360: 39-46 (2010) with minor modifications to restriction enzyme sites. These libraries were panned against biotinylated human PD-1/PDCD1 (Acro Biosystems cat. no. PD1-H82E4) according to art-known panning schemes, for example, as

- 81 043064 is described in WO 2009/085462 and Shi et al., J. Mol. Biol. 397: 385-396 (2010). Phages were obtained using helper phage infection. Binding phages were recovered by adding beads to form a bead/antigen/phage complex. After the last wash, the phage was released by infecting exponentially growing Escherichia coli TG-1 cells.

For subsequent screening, plasmid DNA was prepared from an overnight culture of Escherichia coli TG-1 cells, and the pIX gene was extracted by digestion with NheI/SpeI. After ligation, the DNA was transformed into TG-1 cells and grown on LB/Agar plates overnight. The next day, overnight colonies were harvested and cultures were used:

(i) to perform V region sequencing; and (ii) to induce Fab production.

To obtain Fab, the overnight culture was diluted 100-fold with new medium and grown for 5-6 hours at 37°C. Fab production was induced by adding fresh medium containing IPTG and cultures were grown overnight at 30°C. The next day, cultures were centrifuged and supernatants containing soluble Fab proteins were used for Fab ELISA. For ELISA, soluble Fab proteins were captured onto plates with a polyclonal antibody to Fd(CH1). After washing and blocking, biotinylated human PD1/PDCD1 was added at a concentration of 5 nM. This concentration allows the Fab variants to be ranked according to the fold change relative to the original antibody, with the original Fab present as a control on all plates taken as 100%. Biotinylated human PD-1 was detected with HRP-conjugated streptavidin and chemiluminescence was analyzed in a plate spectrophotometer. By this criterion, 3 heavy and 2 light chains were selected that bind human PD-1 10 or more times stronger than the original Fab.

In table. 20 shows a variant with substitutions at position 57 in HCDR2 from VH and position.

or 30 in LCDR1 from VL. In table. 21 shows the SEQ ID NOs for CDRs of antibodies. In table. 22 shows the SEQ ID NOs for the amino acid sequences of the VH, VL, HC, and LC regions. In table. 23 shows SEQ ID NOs for polynucleotides encoding the VH, VL, HC, and LC regions of antibodies. In table. 24 shows the amino acid sequences of HCDR1, HCDR2 and HCDR3. In table. 25 shows the amino acid sequences of LCDR1, LCDR2 and LCDR3. In table. 26 shows the amino acid sequences of the VH region. In table. 27 shows the amino acid sequences of the VL region. Affinity matured variants are expected to bind the same epitope on PD-1 as the parent PD1B878 antibody.

Table 20

Identifier VH peptide identifier VH mutation compared to the original variant Peptide identifier VL VL mutation compared to the original variant PD1B1085 PD1H585 E57Y PD1L469 PD1B1086 PD1H586 E57H PD1L469 PD1B1087 PD1H587 E57W PD1L469 PD1B1O88 PD1H405 PD1L651 V29F PD1B1089 PD1H405 PD1L652 S30P PD1B1090 PD1H585 E57Y PD1L651 V29F PD1B1091 PD1H586 E57H PD1L651 V29F PD1B1092 PD1H587 E57W PD1L651 V29F PD1B1093 PD1H585 E57Y PD1L652 S30P PD1B1094 PD1H586 E57H PD1L652 S30P PD1B1095 PD1H587 E57W PD1L652 S30P

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Table 21

Antibody HCDR1 HCDR2 HDDR3 LCDR1 LCDR2 LCDR3 PD1B1085 2 145 4 5 6 7 PD1B1086 2 146 4 5 6 7 PD1B1087 2 147 4 5 6 7 PD1B1O88 2 3 4 148 6 7 PD1B1089 2 3 4 149 6 7 PD1B1090 2 145 4 148 6 7 PD1B1091 2 146 4 148 6 7 PD1B1092 2 147 4 148 6 7 PD1B1093 2 145 4 149 6 7 PD1B1094 2 146 4 149 6 7 PD1B1095 2 147 4 149 6 7

Table 22

mAb ID VH peptide identifier Peptide ID VL Vh with SEQ ID NO: VL with SEQ ID NO: NS with SEQ ID NO: LC with SEQ ID NO: PD1B1085 PD1H585 PD1L469 140 16 150 28 PD1B1086 PD1H586 PD1L469 141 16 151 28 PD1B1087 PD1H587 PD1L469 142 16 152 28 PD1B1O88 PD1H405 PD1L651 10 143 22 153 PD1B1089 PD1H405 PD1L652 10 144 22 154 PD1B1090 PD1H585 PD1L651 140 143 150 153 PD1B1091 PD1H586 PD1L651 141 143 151 153 PD1B1092 PD1H587 PD1L651 142 143 152 153 PD1B1093 PD1H585 PD1L652 140 144 150 154 PD1B1094 PD1H586 PD1L652 141 144 151 154 PD1B1095 PD1H587 PD1L652 142 144 152 154

Table 23

mAb ID Name peptide vh Name peptide VL VH cDNA with SEQ ID NO: VL cDNA with SEQ ID NO: HC cDNA with SEQ ID NO: cDNA LC c SEQID NO: PD1B1085 PD1H585 PD1L469 155 19 160 31 PD1B1086 PD1H586 PD1L469 156 19 161 31 PD1B1087 PD1H587 PD1L469 157 19 162 31 PD1B1O88 PD1H405 PD1L651 13 158 25 163 PD1B1089 PD1H405 PD1L652 13 159 25 164 PD IB 1090 PD1H585 PD1L651 155 158 160 163 PD1B1091 PD1H586 PD1L651 156 158 161 163 PD IB 1092 PD1H587 PD1L651 157 158 162 163 PD1B1093 PD1H585 PD1L652 155 159 160 164 PD IB 1094 PD1H586 PD1L652 156 159 161 164 PD1B1095 PD1H587 PD1L652 157 159 162 164

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Table 24

Antibody HCDR1 HCDR2 HDDR3 (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) PD1B1085 GYTFTDYSMH (SEQ ID NO: 2) WINIETGYPT (SEQ ID NO: 145) DYYGTYFYAMDY (SEQ ID NO: 4) PD1B1086 GYTFTDYSMH (SEQ ID NO: 2) WINIETGHPT (SEQ ID NO: 146) DYYGTYFYAMDY (SEQ ID NO: 4) PD1B1087 GYTFTDYSMH (SEQ ID NO: 2) WINIETGWPT (SEQ ID NO: 147) DYYGTYFYAMDY (SEQ ID NO: 4) PD1B1O88 GYTFTDYSMH (SEQ ID NO: 2) WINIETGEPT (SEQ ID NO: 3) DYYGTYFYAMDY (SEQ ID NO: 4) PD1B1089 GYTFTDYSMH (SEQ ID NO: 2) WINIETGEPT (SEQ ID NO: 3) DYYGTYFYAMDY (SEQ ID NO: 4) PD1B1090 GYTFTDYSMH (SEQ ID NO: 2) WINIETGYPT (SEQ ID NO: 145) DYYGTYFYAMDY (SEQ ID NO: 4) PD1B1091 GYTFTDYSMH (SEQ ID NO: 2) WINIETGHPT (SEQ ID NO: 146) DYYGTYFYAMDY (SEQ ID NO: 4) PD1B1092 GYTFTDYSMH (SEQ ID NO: 2) WINIETGWPT (SEQ ID NO: 147) DYYGTYFYAMDY (SEQ ID NO: 4) PD1B1093 GYTFTDYSMH (SEQ ID NO: 2) WINIETGYPT (SEQ ID NO: 145) DYYGTYFYAMDY (SEQ ID NO: 4) PD1B1094 GYTFTDYSMH (SEQ ID NO: 2) WINIETGHPT (SEQ ID NO: 146) DYYGTYFYAMDY (SEQ ID NO: 4) PD1B1095 GYTFTDYSMH (SEQ ID NO: 2) WINIETGWPT (SEQ ID NO: 147) DYYGTYFYAMDY (SEQ ID NO: 4)

Table 25

Antibody LCDR1 (SEQ ID NO:) LCDR2 (SEQ ID NO:) LCDR3 (SEQID NO:) PD1B1085 TASSSVSSSYLH STSNLAS HQYHRSPLT (SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) PD1B1086 TASSSVSSSYLH (SEQ ID NO: 5) STSNLAS (SEQ ID NO: 6) HQYHRSPLT (SEQ ID NO: 7) PD1B1087 TASSSVSSSYLH (SEQ ID NO: 5) STSNLAS (SEQ ID NO: 6) HQYHRSPLT (SEQ ID NO: 7) PD1B1O88 TASSSFSSSYLH (SEQ ID NO: 148) STSNLAS (SEQ ID NO: 6) HQYHRSPLT (SEQ ID NO: 7)

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PD1B1089 TASSSVPSSYLH (SEQ ID NO: 149) STSNLAS (SEQ ID NO: 6) HQYHRSPLT (SEQ ID NO: 7) PD1В1090 TASSSFSSSYLH (SEQ ID NO: 148) STSNLAS (SEQ ID NO: 6) HQYHRSPLT (SEQ ID NO: 7) PD1B1091 TASSSFSSSYLH (SEQ ID NO: 148) STSNLAS (SEQ ID NO: 6) HQYHRSPLT (SEQ ID NO: 7) PD IB 1092 TASSSFSSSYLH (SEQ ID NO: 148) STSNLAS (SEQ ID NO: 6) HQYHRSPLT (SEQ ID NO: 7) PD1B1093 TASSSVPSSYLH (SEQ ID NO: 149) STSNLAS (SEQ ID NO: 6) HQYHRSPLT (SEQ ID NO: 7) PD IB 1094 TASSSVPSSYLH (SEQ ID NO: 149) STSNLAS (SEQ ID NO: 6) HQYHRSPLT (SEQ ID NO: 7) PD1B1095 TASSSVPSSYLH (SEQ ID NO: 149) STSNLAS (SEQ ID NO: 6) HQYHRSPLT (SEQ ID NO: 7)

Table 26

Name peptide VH Amino acid sequence of VH SEQ ID NO: PD1H585 QVQLVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRQAP GQGLEWMGWINIETGYPTYAQGFTGRFVFSLDTSVSTAYLQIS SLKAEDTAVYFCARDYYGTYFYAMDYWGQGTLVTVSS 140 PD1H586 QVQLVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRQAP GQGLEWMGWINIETGHPTYAQGFTGRFVFSLDTSVSTAYLQIS SLKAEDTAVYFCARDYYGTYFYAMDYWGQGTLVTVSS 141 PD1H587 QVQLVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRQAP GQGLEWMGWINIETGWPTYAQGFTGRFVFSLDTSVSTAYLQI SSLKAEDTAVYFCARDYYGTYFYAMDYWGQGTLVTVSS 142

Table 27

Name peptide VL VL amino acid sequence SEQ ID NO: PD1L651 EIVLTQSPATLSLSPGERATLSCTASSSFSSYLHWYQQKPGLAP RLLIYSTSNLASGIPDRFSGSGSGTDYTLTISRLEPEDFAVYYCH QYHRSPLTFGQGTKLEIK 143 PD1L652 EIVLTQSPATLSLSPGERATLSCTASSSVPSSYLHWYQQKPGLA PRLLIYSTSNLASGIPDRFSGSGSGTDYTLTISRLEPEDFAVYYC HQYHRSPLTFGQGTKLEIK 144

-85 043064

SEQ ID NO: 150HC PD1B1085, PD1B1090, PD1B1093

QVQLVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRQAPGQGLEWMGWIN IETGYPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCARDYYGTYFYAMDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 151HC PD1B1086, PD1B1091, PD1B1094

QVQLVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRQAPGQGLEWMGWIN IETGHPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCARDYYGTYFYAMDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 152HC PD1B1087, PD1B1092, PD1B1095

QVQLVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRQAPGQGLEWMGWIN IETGWPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCARDYYGTYFYAMDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 153 LC PD1B1O88, PD1B1090, PD1B1091, PD1B1092

EIVLTQSPATLSLSPGERATLSCTASSSFSSSYLHWYQQKPGLAPRLLIYSTSNLAS GIPDRFSGSGSGTDYTLTISRLEPEDFAVYYCHQYHRSPLTFGQGTKLEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 154 LC PD1B1089, PD1B1093, PD1B1094, PD1B1095

EIVLTQSPATLSLSPGERATLSCTASSSVPSSYLHWYQQKPGLAPRLLIYSTSNLAS GIPDRFSGSGSGTDYTLTISRLEPEDFAVYYCHQYHRSPLTFGQGTKLEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 155 cDNA of PD1H585

CAGGTGCAGCTGGTGCAGTCTGGAAGCGAACTGAAGAAACCTGGAGCCTCTG TGAAAGTGTCTTGTAAGGCCAGCGGCTACACCTTCACCGACTACAGCATGCACTGGG

- 86 043064

TGCGGCAGGCCCCTGGACAGGGCCTGGAATGGATGGGCTGGATCAACATCGAGACC GGCTATCCCACCTACGCCCAGGGCTTTACCGGACGGTTCGTGTTCAGCCTGGATACA GC CAGGGAACACTGGTGACAGTGTCTTCT

SEQ ID NO: 156 cDNA of PD1H586

CAGGTGCAGCTGGTGCAGTCTGGAAGCGAACTGAAGAAACCTGGAGCCTCTG TGAAAGTGTCTTGTAAGGCCAGCGGCTACACCTTCACCGACTACAGCATGCACTGGG TGCGGCAGGCCCCTGGACAGGGCCTGGAATGGATGGGCTGGATCAACATCGAGACC GGCCATCCCACCTACGCCCAGGGCTTTACCGGACGGTTCGTGTTCAAGCCTGGATA CA TCTGTGTCTACAGCCTATCTGCAGATCAGCTCTCTGAAGGCCGAAGATACAGCCGTG TACTTCTGCGCCCGGGACTACTACGGCACCTACTTCTACGCCATGGACTACTGGGGC CAGGGAACACTGGTGACAGTGTCTTCT

SEQ ID NO: 157 cDNA of PD1H587

CAGGTGCAGCTGGTGCAGTCTGGAAGCGAACTGAAGAAACCTGGAGCCTCTG TGAAAGTGTCTTGTAAGGCCAGCGGCTACACCTTCACCGACTACAGCATGCACTGGG TGCGGCAGGCCCCTGGACAGGGCCTGGAATGGATGGGCTGGATCAACATCGAGACC GGCTGGCCCACCTACGCCCAGGGCTTTACCGGACGGTTCGTGTTCAAGCCTGGATA CA TCTGTGTCTACAGCCTATCTGCAGATCAGCTCTCTGAAGGCCGAAGATACAGCCGTG TACTTCTGCGCCCGGGACTACTACGGCACCTACTTCTACGCCATGGACTACTGGGGC CAGGGAACACTGGTGACAGTGTCTTCT

SEQ ID NO: 158 cDNA PD1L651

ACCATCA GCCGGCTGGAACCTGAGGATTTTGCCGTGTACTACTGCCACCAGTACCAC CGGAGCCCCCTGACCTTCGGCCAGGGAACAAAGCTGGAAATCAAG

SEQ ID NO: 159 cDNA PD1L652

ACCATCA GCCGGCTGGAACCTGAGGATTTTGCCGTGTACTACTGCCACCAGTACCAC CGGAGCCCCCTGACCTTCGGCCAGGGAACAAAGCTGGAAATCAAG

SEQ ID NO: 160 cDNA HC PDIB 1085, PDIB 1090, PDIB 1093

TCTGATAAGAGTCAGAGGTAACTCCCGTTGCGGTGCTGTTAACGGTGGAGGG CAGTGTGTAGTCTGAGCAGTACTCGTTGCTGCCGCGCGCCACCAGACATAATAGCTG ACAGACTAACAGACTGTTCCTTTCCATGGTCTTTTCTGCAGTCACCGTCCTTAGATC CACTAGTCCAGTGTGGTGAAGCTTGCCGCCACCATGGCTTGGGTGTGGACCTTGCTA

- 87 043064

TTCCTGATGGCAGCTGCCCAAAGTATACAGGCCCAGGTGCAGCTGGTGCAGTCTGG AAGCGAACTGAAGAAACCTGGAGCCTCTGTGAAAGTGTCTTGTAAGGCCAGCGGCT ACACCTTCACCGACTACAGCATGCACTGGTGCGGCAGGCCCCTGGACAGGGCCTG GAATGGATGGGCTGGATCAACATCGAGACCGGCTATCCCACCTACGCCCAGGGCTT T ACCGGACGGTTCGTGTTCAGCCTGGATACATCTGTGTCTACAGCCTATCTGCAGAT CAGCTCTCTGAAGGCCGAAGATACAGCCGTGTACTTCTGCGCCCGGGACTACTACGG CACCTACTTCTACGCCATGGACTACTGGGCCAGGGAACACTGGTGACAGTGTCTTC TGCCTCCACCAAGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTC TGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGA CGGTGTCGTGGAACTCAGGCGCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCC TACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCT TGGGCACCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGG TG GACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCC AGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA TG CCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTC CTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTC CAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGC CCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAAC CAGGTCAGCCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAG TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA AC GCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGATAGTTCGAATTCCTAGAAGACAT GATAAGATACATTGATGAGTTTGGACAAAACCACAACTAGAATGCAGTGAAAAAAAT GCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAA TAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGT GT GGGA

SEQ ID NO: 161 cDNA HC PDIB 1086, PDIB 1091, PDIB 1094

TCTGATAAGAGTCAGGTAACTCCCGTTGCGGTGCTGTTAACGGTGGAGGG CAGTGTGTAGTCTGAGCAGTACTCGTTGCTGCCGCGCGCCACCAGACATAATAGCTG ACAGACTAACAGACTGTTCCTTTCCATGGTCTTTTCTGCAGTCACCGTCCTTAGATC CACTAGTCCAGTGTGGTGAAGCTTGCCGCCACCATGGCTTGGGTGTGGACCTTGCTA TTC CTGATGGCAGCTGCCCAAAGTATACAGGCCCAGGTGCAGCTGGTGCAGTCTGG AAGCGAACTGAAGAAACCTGGAGCCTCTGTGAAAGTGTCTTGTAAGGCCAGCGGCT ACACCTTCACCGACTACAGCATGCACTGGGTGCGGCAGGCCCCTGGACAGGGCCTG GAATGGATGGGCTGGATCAACATCGAGACCGGCCATCCCACCTACGCCCAGGGCTT TACCGGA CGGTTCGTGTTCAGCCTGGATACATCTGTGTCTACAGCCTATCTGCAGAT CAGCTCTCTGAAGGCCGAAGATACAGCCGTGTACTTCTGCGCCCGGGACTACTACGG

- 88 043064

CACCTACTTCTACGCCATGGGACTACTGGGGCCAGGGAACACTGGTGACAGTGTCTTC TGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCTCCTCCAAGAGCACCTC CCCGGCTGTCC TACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGTGTGACCGTGCCCTCCAGCAGCT TGGGCACCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTG GACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCC AGACCTGAACTCCTGGGGGACCGTCAGTCTTCCTCTTCCCCAAAACC CAAGGA CACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCA CGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGTGCATAATG CCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTC CTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTC CAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGC CCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAAC CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAG TGCTGGA CTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCA GCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCCTCTGCACAACCACTACAC GCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGATAGTTCGAATTCCTAGAAGACAT GATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAA AT GCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAA TAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGT GTGGGA

SEQ ID NO: 162 cDNA HC PDIB 1087, PDIB 1092, PDIB 1095

TCTGATAAGAGTCAGGTAACTCCCGTTGCGGTGCTGTTAACGGTGGAGGG CAGTGTGTAGTCTGAGCAGTACTCGTTGCTGCCGCGCGCCACCAGACATAATAGCTG ACAGACTAACAGACTGTTCCTTTCCATGGTCTTTTCTGCAGTCACCGTCCTTAGATC CACTAGTCCAGTGTGGTGAAGCTTGCCGCCACCATGGCTTGGGTGTGGACCTTGCTA TTC CTGATGGCAGCTGCCCAAAGTATACAGGCCCAGGTGCAGCTGGTGCAGTCTGG AAGCGAACTGAAGAAACCTGGAGCCTCTGTGAAAGTGTCTTGTAAGGCCAGCGGCT ACACCTTCACCGACTACAGCATGCACTGGGTGCGGCAGGCCCCTGGACAGGGCCTG GAATGGATGGGCTGGATCAACATCGAGACCGGCTGGCCCACCTACGCCCAGGGCTT TACCGGA CGGTTCGTGTTCAGCCTGGATACATCTGTGTCTACAGCCTATCTGCAGAT CAGCTCTCTGAAGGCCGAAGATACAGCCGTGTACTTCTGCGCCCGGGACTACTACGG CACCTACTTCTACGCCATGGACTACTGGGCCAGGGAACACTGGTGACAGTGTCTTC TGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCCTCCTCCAAGAGCACCTC TGGG GGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGA CGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCC TACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGTGTGACCGTGCCCTCCAGCAAGCT TGGGCACCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTG

- 89 043064

GACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCC AGCACCTGAACTCCTGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA TG CCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTC CTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTC CAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGC CCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAAC CAGGTCAGCCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAG TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA AC GCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGATAGTTCGAATTCCTAGAAGACAT GATAAGATACATTGATGAGTTTGGACAAAACCACAACTAGAATGCAGTGAAAAAAAT GCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAA TAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGT GT GGGA

SEQ ID NO: 163 cDNA LC PDIB 1088, PDIB 1090, PDIB 1091, PDIB 1092

TCTGATAAGAGTCAGGTAACTCCCGTTGCGGTGCTGTTAACGGTGGAGGG CAGTGTGTAGTCTGAGCAGTACTCGTTGCTGCCGCGCGCCACCAGACATAATAGCTG ACAGACTAACAGACTGTTCCTTTCCATGGTCTTTTCTGCAGTCACCGTCCTTAGATC CACTAGTCCAGTGTGGTGAAGCTTGCCGCCACCATGGCTTGGGTGTGGACCTTGCTA TTC CTGATGGCGGCCGCCCAAAGTATACAGGCCGAGATCGTGCTGACACAGTCTCCT GCCACACTGTCTCTGTCTCCTGGAGAACGGGCCACACTGAGCTGCACCGCCAGCAGC AGCGGATCTGGCACAGATTACACACTGACCATCAGCCGGCTGGAACCTGAGGATT TTGCCGTGTACTACTGCCACCAGTACCACCGGAGCCCCCTGACCTTCGGCCAGGGAA CAAAGCTGGAAATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCAT CTGATGAGCAGTTGAAATCTGGAACTGCTCTGTTGTGTGCCTGCTGAATAACTTCT ATCCCAGAG AGGCCAAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAAC TCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAG CACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAG TCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT TAGTGATTC GAATTCCTAGAAGACATGATAAGATACATTGATGAGTTTGGACAAACC ACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCT TTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCAT TTTATGTTTCAGGTTCAGGGGGAGTGTGTGGA

SEQ ID NO: 164 cDNA LC PD1B1089, PD1B1093, PD1B1094, PD1B1095

TCTGATAAGAGTCAGAGGTAACTCCCGTTGCGGTGCTGTTAACGGTGGAGGG

- 90 043064

CAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGCGCGCGCCACCAGACATAATAGCTG

ACAGACTAACAGACTGTTCCTTTCCATGGGTCTTTTCTGCAGTCACCGTCCTTAGATC CACTAGTCCAGTGTGGTGAAGCTTGCCGCCACCATGGCTTGGGTGTGGACCTTGCTA TTCCTGATGGCGGCCGCCCAAAGTATACAGGCCGAGATCGTGCTGACACAGTCTCCT GCCACACTGTCTCTGTCTCCTGGAGAACGGGCCACACTGAGCTGCACCGCCAGCA GC AGCGTGCCAAGCAGCTACCTGCACTGGTACCAGCAGAAACCTGGACTGGCCCCTCG GCTGCTGATCTACAGCACCAGCAACCTGGCCAGCGGCATCCCTGATCGGTTTTCTGG CAGCGGATCTGGCACAGATTACACACTGACCATCAGCCGGCTGGAACCTGAGGATT TTGCCGTGTACTACTGCCACCAGTACCCGGAGCCCCCTGACCTTCGGCCAGGGAA CAAAGCTGGAAATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCAT CTGATGAGCAGTTGAAATCTGGAACTGCTCTGTTGTGTGTGCCTGCTGAATAACTTCT ATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAAC TCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAG CAC CCTGACGCTGAGCAAAGCAGACTACGAGAAAACACAAAGTCTACGCCTGCGAAG TCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT TAGTGATTCGAATTCCTAGAAGACATGATAAGATACATTGATGAGTTTGGACAAACC ACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCT TTATTTGTAAC CATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCAT TTTATGTTTCAGGTTCAGGGGGAGGTGTGGGA

The affinity of antibodies to human and cynomolgus PD-1 was measured using SPR as described in Example 1. Antibodies bound human PD-1 with a K _D value ranging from 1 x 10 ^-8 M to 10 x 10' ¹⁰ M (Table 28) and PD -1 cynomolgus macaque with a K _D value ranging from 7 x 10' ⁸ M to 1 x 10' ⁹ M (Table 26). The affinities of most affinity matured variants of PD1B878 improved approximately 100-fold over the parent antibody.

Table 28 __________________________________________________________________________

wal Ka (1/Ms) Kd (1/s) KD (M) PD1B1085 1.56E+05 1D2E-04 9D1E- 10 PD1B1086 1.24E+05 3.94E - 04 ZD9E-09 PD1B1087 1.66E+05 2.39E - 04 1D4E-09 PD1B1088 ZDZE+04 3.41Е-04 1.09Е-08 PD1B1089 5.86E+04 4.74E - 04 8.09E - 09 PD1B1090 1.06E+05 2.37E - 05 2.23E - 10 PD1B1091 7.77E+04 6.44E - 05 8,ZOE - 10 PD1B1092 1.40E+05 3.00E - 05 2D4E- 10 PD1B1093 1.25E+05 4.36E - 05 3.49E - 10 PD1B1094 1.05E+05 9D6E-05 8.71E-10 PD1B1095 1.51E+05 7D2E-05 4.71E-10

Table 29

mAb Ka(l/Mc) Kd (1/c) KD(M) PD1B1085 2.00E+05 l,29E-03 6.45E - 09 PD1B1086 1J1E+05 5.44E-03 ZD8E-08 PD1B1087 2D2E+05 2.47E-03 1D6E-08 PD1B1088 6.61E+04 4.79E-03 7.24E - 08 PD1B1089 9.32E+04 8.39E-03 9.00E - 08 PD1В1090 1.3OE+O5 l,85E-04 l,43E-09 PD1B1091 9.91E+04 9.24E-04 9.32E - 09 PD IB 1092 l,29E+05 4D1E-04 ZD8E-09 PD1B1093 l,69E+05 3.68E-04 2D8E-09 PD IB 1094 l,35E+05 l,62E-03 l,20E-08 PD1B1095 l,85E+05 7.81E-04 4.23E-09

Antibodies PDIB1086, PDIB1090 and PDIB1094 were characterized by their ability to inhibit activated T cells in a secondary CMV-specific response assay (CMV-PBMC assay described in Example 1). In table. 30 shows the average percent inhibition for two donors in one experiment. All antibodies tested inhibited the CMV-specific secondary response by 70% or more in the assay.

Table 30

PD1B1086 PD IB 1090 PD1B1094 IgGl Average % 72.7 74.8 77.9 6.0

Example 9 Anti-PD-1 agonist antibodies selectively target chronically activated memory T cells.

It was found that PD-1 was mainly expressed on memory T cells (cells

CD45RO+), but not on intact T cells, and was found to be overexpressed upon T cell activation. PD-1 expression was upregulated in memory T cells stimulated with CMV peptides (Fig. 8A).

Antibodies PD1B849 and PD1B878 were tested for their ability to inhibit the proliferation of activated CD4+ or CD8+ memory T cells using CMV-activated PBMCs. Both PD1B849 and PD1B878 inhibited the proliferation of CDV-specific activated PBMCs in the CMV-PBMC secondary response assay (FIG. 8B).

Example 10 Anti-PD-1 agonistic antibodies reduce the number of activated but non-resting memory T cells by ADCC.

Antibodies PD1B849 and PD1B878 were tested for the ability to mediate ADCC for activated memory T cells or resting memory T cells using NK or PBMC as effector cells. Activated memory T cells were found to have higher expression of PD-1 compared to resting memory T cells. The experiment was carried out according to the protocol described in Example 1. PD1B849 and PD1B878 were expressed in two separate CHO cell lines, with one line producing antibodies with a normal CHO glycosylation profile, and the other producing antibodies having a reduced fucosyl carbohydrate content (for example, low fucose ( LF) cell line). Antibodies expressed in the low-fucose cell line had a fucosyl content of about 1-15%.

PD1B849 and PD1B878 induced ADCC in activated memory T cells in both the presence of NK as effector cells (Fig. 9A, left panel) and PBMCs (Fig. 9A, right panel). Low fucose antibodies (PD1B849-LF and PD1B878-LF) showed stronger ADCC activity against memory T cells. PD1B849 and PD1B878 were unable to induce detectable ADCC in resting T cells that express low levels of PD-1 in the presence of both NK (Fig. 10A, left panel) and PBMC (Fig. 10A, right panel) as effector cells. . PD1B849-LF and PD1B878-LF induced low levels of ADCC in the presence of NK cells or PBMCs.

Example 11 Anti-PD-1 agonist antibodies do not cause CDC.

PD1B849 and PD1B878 were tested for their ability to mediate CDC for activated total T cells with rabbit complement. Activated T cells have higher PD-1 expression compared to resting T cells. The experiment was carried out according to the protocol described in Example 1. PD1B849 and PD1B878 did not induce CDC in activated T cells at the concentrations tested (FIG. 11). The OKT3 positive control showed CDC activity on activated T cells in the presence but not in the absence of complement.

Example 12 Affinity matured antibodies do not block PD-1 binding to PD-L1.

Selected antibodies were tested for their ability to block the binding of dextramerized PD-L1-Fc to PD-1 expressing Jurkat cells. The experiment was carried out according to the protocol described below. PD1B878, PD1B1090 and PD1B1094 did not block PD-L1 binding at the concentrations tested (FIG. 12). As a positive control, a known antibody antagonist was shown that competes with PD-L1 for binding to PD-1 in a dose-dependent manner.

Way. Biotinylated PD-L1-Fc (Acro Biosystems) and SA and APC conjugated dextramers (Immudex) were prepared at a fourfold concentration and mixed at 100 nM:10 nM in staining buffer. To obtain negative controls and non-specific binding controls, mixtures of biotinylated IgG1-Fc with dextramers and only dextramers with medium were prepared in the same way. This mixture was covered with foil and incubated on ice for one hour in preparation for the rest of the experiment. Serial dilutions of test antibodies were prepared in staining buffer at 2-fold concentrations of 20, 2, and 0.2 nM. Jurkat cells expressing PD-1 were harvested on the day of analysis and washed once with staining buffer (BD Pharmingen) by centrifuging the cells at 300 g for 5 min at 4°C. Cells were counted, checked for viability and resuspended at a concentration of 2x10 ⁶ cells/ml in staining buffer. Cells were added at 25 µl/well (50,000 cells/well) to a 96-well U-bottom assay plate, followed by the addition of prepared test antibodies at 50 µl/well. Cells with antibodies were incubated on ice for 15 min. Pre-mixed complexes PD-L1-Fc:dextramer, biotinylated IgG ₁ -Fc:dextramer and dextramers:buffer were added at 25 μl per well. This mixture of cells, antibodies and dextramer-bound PD-L1 was incubated on ice for 1 hour and covered with foil.

The cells were washed twice by adding 150 μl of staining buffer, centrifuged at 300 g for 5 min to pellet the cells, and the plates were shaken to remove the supernatant. The cell pellet from the last wash was resuspended in 40 µl of IntelliCyt running buffer (BD staining buffer with 1 mM EDTA and 0.1% pluronic acid) containing a 1:1000 dilution of Sytox green live/dead cell viability green dye (ThermoFisher) . The final antibody concentrations in the assay were 10, 1, and 0.1 nM. The final concentration of biotinylated PD-L1-Fc ligand protein was 25 nM. The final concentration of dex-92 043064 ramer was 2.5 nM.

The plates were analyzed on an iQue Screener (IntelliCyt). Briefly, cells were gated on FCS/SCS to remove debris. Singlets were gated on SCS-A/SCS-H and live cells were gated from the population of singlets in the low BL1 channel as negative for Sytox green staining for viability. The percentage of positive live cells binding to PD-L1-Fc-dextramer complexes was estimated from the geometric mean on the RL1/APC channel and compared to a negative control, binding to dextramer-Fc complex only. Using advanced metrics in the ForeCyt software (IntelliCyt software), the PD-1 positive population was calculated as % of the live cell population. The percentage specific binding to PD-L1 was calculated as follows:

(% positive with mAb-% positive with IgG1-Fc biotin dextramer complex)/(% positive with isotype control-% positive with IgG1-Fc biotin dextramer complex)x100.

The final results are tabulated in Excel and plotted in Prism, showing % PD-L1 ligand binding in the presence of anti-PD-1 mAb.

Example 13 Anti-PD-1 agonist antibodies are effective in a mouse model of graft versus host disease (GvHD).

To study the effect of PD-1 mAb agonists on pathogenic T cells in vivo, a xenogeneic graft-versus-host (Xeno-GVHD) model was developed by adoptive transfer of human peripheral blood mononuclear cells (PBMCs) to NOD-scid IL-2Ry™ ^u mice. (NSG) with immune deficiency. Female NSG mice (7-9 weeks old) were obtained from Jackson Labs. Before use, the mice were quarantined in a vivarium for one week. Frozen buffy coat isolated human PBMCs were obtained from AllCells, Alameda, California, USA. Before injection, frozen cells were rapidly thawed at 37°C in a water bath and washed 3 times with sterile phosphate-buffered saline (PBS) by centrifugation at 500 g for 5 min at room temperature. The cells were suspended in cold PBS to give a final cell concentration of 50 x 10 ⁶ /ml.

Animals were randomized by body weight and divided into different treatment groups (K=10/group). After randomization, conscious and free-roaming mice received 100 rad (1 Gy) whole body irradiation with a Gammacell® 3000 Elan Irradiator (9.72 Gy/min). Mice were returned to their cages where they were kept.

Each mouse received 25x10 ⁶ human PBMCs in a volume of 500 μl intraperitoneally. Clinical scores and body weight of each animal were recorded daily according to Table 1. 31. Mice that lost >20% of body weight were sacrificed and their clinical endpoint scores recorded according to IACUC guidelines. All animals were euthanized on day 21. Spleens were harvested for analysis by cytometry by fluorescence activated cell sorting (FACS). Skin and colon tissues were collected for histological analysis.

___________________________________________________________ Table 31

Description Clinical scores The usual state of alertness and reactions 0 Tousled coat, decreased activity, eye discharge 1 Slouched posture, moderate hypothermia or hyperthermia, difficulty breathing when prodded 2 Difficulty breathing at rest, ataxia, tremor, hypothermia or hyperthermia 3 Loss of ability to move when pushed, unconsciousness 4 Death 5

Mice were intraperitoneally injected with 10 mg/kg mAbs PD1B505, PD1B506, PD1B849, and PD1B878, which were cloned as mIgG2a chimeric antibodies, as well as anti-PD-1 mAb antagonists (PD1B786 on mouse effector inactive Fc) on a twice daily 4 days/twice schedule. per day 3 days (introduction on days 0, 4, 7, 11, 14 and 18). CTLA-4-Ig, which was used as a control, was administered twice daily for 3 days (day 0, 3, 6, 9, 12, 15 and 18) at 10 mg/kg ip.

After completion of the study, the spleens were removed into cold RPMI1640 medium. After rubbing the spleens through a 70 µm filter and pelleting the cells by centrifugation at 1200 rpm for 5 min at 4°C, the erythrocytes were lysed in ACK lysis buffer (Lonza) on ice for 5 min and

- 93 043064 were washed multiple times in cytometry buffer by fluorescence activated cell sorting (FACS) (PBS/0.5% BSA/2 mM EDTA). Splenocyte viability was assessed by staining cells with ef506 viability dye (eBioscience) according to the manufacturer's instructions. Splenocytes were incubated with human and mouse Fc blocker (BD Biosciences) on ice for 15 min, then stained with optimal concentrations of fluorochrome-conjugated mAbs (1x106 cells in 100 µl of Brilliant Stain buffer) in U-bottomed microtiter plates at 4°C for 30 min and fixed with Fixation Buffer (BD Biosciences). Count Brite pellets (Thermofisher) were added to each sample. FMO controls (fluorescence minus one) were prepared as negative controls for each fluorochrome. Samples were analyzed on an LSRII instrument (BD Biosciences). The following mAbs were used for regulatory T cell assay: anti-human CD45/peridinin-chlorophyll-alpha protein (PCP, clone 2D1), anti-FoxP3/allophycocyanin (APC, clone pCH101), anti-human CD3/allophycocyanin 7 (APCCy7) , clone HIT3a), anti-human CD4/Brilliant Violet 605 (BV605, clone OKT4) and anti-human CD25/Brilliant violet 650 (BV650, clone M-A251). Regulatory T cells were defined as hCD45 ⁺ hCD3 ⁺ hCD4 ⁺ , Foxp3 ⁺ CD25 ⁺ .

In FIG. 13A shows that treatment with PD1B505-mIgG2a and PD1B506-mIgG2a prevented disease progression in a GvHD mouse model. In FIG. 13B shows that PD1B505-mIgG2a and PD1B506-mIgG2a treatment prevented weight loss in the GvHD mouse model. In FIG. 14A shows that treatment with PD1B849-mIgG2a and PD1B878-mIgG2a prevented disease progression in a GvHD mouse model. In FIG. 14B shows that PD1B849-mIgG2a and PD1B878-mIgG2a treatment prevented weight loss in a GvHD mouse model. The reduction in weight loss and clinical scores was associated with an increase in the number of regulatory T cells in the spleen. In FIG. 15 shows the incidence of Treg in the spleen of animals treated with PD1B849-mIgG2a or PD1B878-mIgG2a.

Example 14 Anti-PD-1 agonistic antibodies destroy follicular T helpers (T _FH ) and peripheral T helpers (T _PH ).

Human PBMCs were removed from liquid nitrogen cryostorage and rapidly thawed in a water bath at 37°C until completely thawed. The contents of the vial were transferred to a sterile 50 ml conical tube (separate tubes were used for each donor) and complete RPMI medium (10% FBS, 1x penicillin/streptomycin, 1x sodium pyruvate) was added dropwise to each tube to a total volume of 15 ml. The cells were centrifuged at 250 g for 10 min at room temperature, then the supernatant was removed and the cells were resuspended in 5-10 ml of complete medium and counted using trypan blue displacement. Cells were resuspended at a concentration of 2.5x106 cells/ml and seeded at 2.5x105 cells/well (=100 μl per well) in triplicate in a 96-well U-bottom sterile polystyrene plate. mAb to PD-1 or isotype control human IgG1 - diluted to four times the final concentration and added 50 μl/well to the appropriate wells. After the addition of antibodies, healthy human serum was added to a final concentration of 5%. The total volume in each well was 200 μl, and the cells were incubated for 96 h at 37°C, 5% CO ₂ . In addition to the samples, cells were applied to several additional wells, 5% human serum was added and incubated with the treated samples as controls for flow cytometry staining.

After incubation, the plates were centrifuged at 350 g for 5 min, and air was evacuated from the supernatants. Cells were resuspended in 200 µl PBS, the contents of the repeat wells were pooled and transferred to a new 96-well U-bottom plate for flow cytometry staining. The pooled cells were centrifuged at 350 g for 5 min, air was evacuated from the supernatants, and a fluorescent antibody cocktail was added to each well with the sample (the table of antibodies used is given below). Additional control cells were stained with FMO cocktails to identify critical markers such as but not limited to PD-1, CXCR5, ICOS for use in the gating assay. Cells were stained for 25 min in the dark at room temperature and then centrifuged at 350 g for 5 min. The cells were washed twice in 200 µl of PBS, after which 100 µl of 4% paraformaldehyde was added to each well to fix the cells. Cells were fixed for 10 min in the dark at 4°C, then washed once with PBS+1% BSA and resuspended in 200 μl PBS+1% BSA. Counting beads (Invitrogen) were added at 5 μl/well (5000 beads) per sample and samples were analyzed on a BD LSRII flow cytometer. Flow cytometry data were analyzed using FlowJo software. Sample counts were normalized by the number of beads as follows: number of cells per sample=(number of cells counted x 5000 beads added)/(number of beads counted). Samples were normalized for isotype control - huIgG1 - as follows:

(bead-normalized number of sample cells/bead-normalized number of isotype cells) x 100 and expressed as a percentage (%).

Graphs were built for the data using the GraphPad Prism software (version 7).

The following cell populations were described during the analysis:

- 94 043064 follicular T helpers (T _F H): live, CD19 ^- CD56 ^- /CD4 ⁺ CD45RO ⁺ /HLADR ⁺ /CXCR5 ⁺ /ICOS ⁺ PD1 ⁺ ;

peripheral T-helpers (T _PH ): live, CD19 ^- CD567CD4 ⁺ CD45RO ⁺ /HLADR ⁺ /CXCR57ICOS ⁺ PD1 ⁺ ;

combined TFH/TPH population: live, CD19'CD567CD4 ⁺ CD45RO ⁺ /HLADR ⁺ /ICOS ⁺ PD1 ⁺ .

In FIG. 16 shows a dose-response curve for T _FH /T _PH pooled population reduction mediated by antibodies PD1B878, PD1B878-FL (low fucose), PD1B1090, and PD1B1094. Data are presented as mean % change in T _fh /T _ph cell numbers relative to isotype control using n=8 healthy human donors (n=7 for PD1B1O9O). The PD1B878-FL antibody most effectively reduced the T _FH /T _PH population.

Claims (25)

1. An isolated antibody that specifically binds to PD-1, or an antigen-binding fragment thereof, comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3 with SEQ ID sequences NO: 2, 3, 4, 5, 6 and 7, respectively. 2. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment has one, two, three, four or five of the following properties: a) do not block PD-L1 binding to PD-1; moreover, the absence of blocking is measured by the inability of the antibody to inhibit the clustering of cells expressing PD-L1 and cells expressing PD-1, as described in example 1; b) bind to PD-1 with an equilibrium dissociation constant (K _D ) of about 5x108M or less, with KD being measured with a ProteOn XPR36 system at +25°C; c) bind to PD-1 with an association constant (Ka) of about 3x10 ⁴ 1/Ms or more, with Ka being measured with a ProteOn XPR36 system at +25°C; d) bind to PD-1 with a dissociation constant (Kd) of about 3x10 ^-3 1/s or less, and Kd is measured using the ProteOn XPR36 system at +25°C; or e) inhibit the proliferation of antigen-specific T cells; while proliferation is assessed in the CMV-PBMC assay as described in Example 1. 3. An antibody or antigen-binding fragment according to claim 1 or 2, containing: a) heavy chain variable region (VH) framework derived from IGHV7-4-1*1 (SEQ ID NO: 125); b) light chain variable region framework (VL) derived from IGKV3D-20*1 (SEQ ID NO: 126); or c) a VH framework derived from IGHV7-4-1*1 (SEQ ID NO: 125) and a VL framework derived from IGKV3D20*1 (SEQ ID NO: 126). 4. An antibody or antigen-binding fragment according to any one of claims 1-3, containing: a) heavy chain variable region (VH) of SEQ ID NO: 10; b) light chain variable region (VL) of SEQ ID NO: 16; or c) VH and VL with SEQ ID NO: 10 and 16, respectively. 5. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof: a) is a PD-1 agonist; b) mediates antibody-dependent cellular cytotoxicity (ADCC) of cells expressing PD-1; or c) is a PD-1 agonist and mediates ADCC of cells expressing PD-1. 6. An antibody or antigen-binding fragment thereof according to claim 5, wherein the cells expressing PD-1 are activated memory T cells, follicular helper T cells (TFH), peripheral helper T cells (TPH), or any combination thereof. 7. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof is an IgG1, IgG ₂ , IgG ₃ or IgG4 isotype. 8. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof contains at least one mutation in the Fc of the antibody that modulates the binding of the antibody to the Fc receptor (FcR). 9. An antibody or antigen-binding fragment thereof according to claim 8, wherein at least one mutation is: a) S267E mutation; b) mutation S267D; c) mutation S267E/I332E; d) S267E/L328F mutation; e) mutation G236D/S267E; f) P238D mutation; or g) P238D/E233D/G237D/H268D/P271G/A330R mutation. 10. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody - 95 043064 contains: a) heavy chain (HC) with SEQ ID NO: 22; b) light chain (LC) of SEQ ID NO: 28; or c) HC with SEQ ID NO: 22 and LC with SEQ ID NO: 28. 11. An antibody or antigen-binding fragment thereof according to any one of claims 1-10, wherein the antibody or antigen-binding fragment thereof has a 2-antennary glycan structure with a fucose content ranging from about 1% to about 15%. 12. A polynucleotide containing a nucleic acid sequence encoding an antibody or antigen-binding fragment according to any one of claims 1-11. 13. A vector containing at least one polynucleotide according to claim 12. 14. A host cell containing the vector of claim 13. 15. A host cell according to claim 14, which is a eukaryotic cell, a prokaryotic cell, a CHO cell, a HEK293 cell, or a hybridoma. 16. A method for producing an antibody or antigen-binding fragment thereof, comprising culturing a host cell according to claim 14 under conditions under which the antibody or antigen-binding fragment is expressed, and isolating the antibody or antigen-binding fragment thereof. 17. Pharmaceutical composition containing: a) an antibody or antigen-binding fragment according to any one of claims 1-11; And b) a pharmaceutically acceptable carrier. 18. A method for suppressing the activation of a PD-1 expressing T cell in a patient, comprising administering to the patient the isolated antibody or an antigen-binding fragment thereof according to any one of claims 1 to 11 for a time sufficient to suppress the activation of a PD-1 expressing T cell . 19. The method of claim 18 wherein the PD-1 expressing T cell is a CD4+ antigen specific T cell and/or a CD8+ antigen specific T cell. 20. A method for reducing an immune response, comprising administering to a patient in need thereof a therapeutically effective amount of an isolated antibody or an antigen-binding fragment thereof according to any one of claims 1-11. 21. A method of treating an immune disorder, comprising administering to a patient in need thereof a therapeutically effective amount of the isolated antibody or antigen-binding fragment thereof according to any one of claims 1-11. 22. The method of claim 21 wherein the immune disorder is lupus, systemic lupus erythematosus, Sjögren's syndrome, arthritis, rheumatoid arthritis, asthma, COPD, pelvic inflammatory disease, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, psoriatic arthritis, vasculitis, surgical adhesions, stroke, type I diabetes, Lyme disease, meningoencephalitis, autoimmune uveitis, multiple sclerosis, Guillain-Barré syndrome, atopic dermatitis, autoimmune hepatitis, fibrosizing alveolitis, Basedov disease due to Iga nephropathy, idiopathic thrombocytopenic purple, Meniere disease, pemucrchis, primary biliary cirrhosis, sarcoidosis, scleroderma, granulomatosis of the vegener, other autoimmune disorders, pancreatitis, reaction of the transplant against the host, jerk the transplant, infertility associated with lack of tolerance of the fetus to the mother, Sjögren's syndrome, vitiligo, myasthenia gravis or systemic scleroderma. 23. The method of any one of claims 18-22, wherein the antibody, or antigen-binding fragment thereof, is administered in combination with a second therapeutic agent. 24. An immunoconjugate containing an antibody or antigen-binding fragment according to any one of claims 1 to 11 conjugated to a detectable label. 25. An immunoconjugate comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 conjugated to a cytotoxic agent.