Anti-pd-1 monoclonal antibodies and methods of making the same

Abstract

Disclosed herein is an anti-human PD-1 monoclonal antibody and a preparation method thereof. The anti-human PD-1 monoclonal antibody can bind to an extracellular region of a human PD-1 protein receptor, and can block and block the binding of the PD-1 protein at the protein level and the cell level, thereby enhancing immunity. The monoclonal antibody can be independently administered, administered in combination with other drugs, and / or administered independently or in any given order in combination, to treat individuals in need thereof.

Description

[0001] sequence list This application was filed electronically via EFS-Web and includes a sequence listing submitted electronically in ST.26 format as an .xml file. The .xml file contains a sequence listing named “025-PCT-SEQ-Listing.xml”, generated on November 8, 2024, and has a size of 23,068 bytes on disk and 24,576 bytes in length. The sequence listing contained in this .xml file is part of this specification and is incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure relates to the biomedical field, and more particularly to anti-PD-1 monoclonal antibodies optionally combined with another therapeutic agent (e.g., pegylated human interferon α2b), as well as methods for their preparation and applications. Background Technology

[0003] Programmed protein death 1 (PD-1) is known in the field. Simply put, it is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, macrophages, and myeloid cells. The initial members of this family, CD28 and ICOS, were discovered through their functional enhancement of T cell proliferation following the addition of monoclonal antibodies.

[0004] The PD-1 gene is a 50-55 kDa type I transmembrane protein and part of the Ig gene superfamily. PD-1 contains a proximal immunoreceptor tyrosine inhibitory motif (ITIM) and a distal tyrosine-based switch motif (ITSM). Although structurally similar to CTLA-4, PD-1 lacks the MYPPPY motif, which is crucial for the binding of B7-1 and B7-2. Two ligands for PD-1 have been identified. PD-L1 and PD-L2 have been shown to downregulate T cell activation upon binding to PD-1. Both PD-L1 and PD-L2 are B7 homologs; they bind to PD-1 but not to other CD28 family members. One ligand of PD-1, PD-L1, is abundant in various human cancers. The interaction between PD-1 and PD-L1 leads to a reduction in tumor-infiltrating lymphocytes, T cell receptor-mediated proliferation, and immune evasion by cancer cells. Inhibiting the local interaction between PD-1 and PD-L1 can reverse immunosuppression, and the effects are additive when the interaction between PD-1 and PD-L2 is blocked simultaneously. PD-1 has been found to play a role in autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease (GVHD), type 1 diabetes, and rheumatoid arthritis. Summary of the Invention

[0005] In a first aspect, this disclosure relates to an isolated antibody or antigen-binding portion thereof that preferentially binds to PD-1, comprising: a heavy chain complementarity-determining region 1 (VHCDR1) comprising the amino acid sequence listed in SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 21, a heavy chain complementarity-determining region 2 (VHCDR2) comprising the amino acid sequence listed in SEQ ID NO: 10, and / or a heavy chain complementarity-determining region 3 (VHCDR3) comprising the amino acid sequence listed in SEQ ID NO: 11 or SEQ ID NO: 20; and a light chain complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence listed in SEQ ID NO: 12, a light chain complementarity-determining region 2 (VLCDR2) comprising the amino acid sequence listed in SEQ ID NO: 13, and / or a light chain complementarity-determining region 3 (VLCDR3) comprising the amino acid sequence listed in SEQ ID NO: 14.

[0006] In a second aspect, this disclosure relates to an isolated antibody or antigen-binding portion thereof that preferentially binds to PD-1, comprising: a heavy chain variable region (VH) comprising SEQ ID NO: 5 or SEQ ID NO: 22; and a light chain variable region (VL) comprising SEQ ID NO: 6.

[0007] In another aspect, this disclosure relates to an isolated antibody or its antigen-binding portion that preferentially binds to PD-1, comprising: a heavy chain complementarity-determining region 1 (VHCDR1), a heavy chain complementarity-determining region 2 (VHCDR2), and / or a heavy chain complementarity-determining region 3 (VHCDR3) of the heavy chain variable region amino acid sequence listed in SEQ ID NO: 5 or obtained from (e.g., determined, derived, or copied therefrom) the heavy chain variable region amino acid sequence; and a light chain complementarity-determining region 1 (VLCDR1), a light chain complementarity-determining region 2 (VLCDR2), and / or a light chain complementarity-determining region 3 (VLCDR3) of the light chain variable region amino acid sequence listed in SEQ ID NO: 6 (e.g., determined, derived, or copied therefrom).

[0008] Other aspects include the fact that the isolated antibody or its antigen-binding portion that preferentially binds to PD-1 further comprises a heavy chain constant region (HCCR) comprising the amino acid sequence listed in SEQ ID NO: 7; and wherein the HCCR optionally comprises one, two or three conserved amino acid substitutions.

[0009] The additional aspect includes the isolated antibody or its antigen-binding portion of aspect 2, which further comprises a light chain constant region (LCCR) comprising the amino acid sequence of SEQ ID NO: 8; and wherein the LCCR optionally comprises one, two or three conserved modified amino acid substitutions.

[0010] Other aspects of aspect 2 include the isolated antibody or antigen-binding portion thereof of aspect 2, which further comprises a heavy chain constant region (HCCR) comprising the amino acid sequence listed in SEQ ID NO: 7, wherein the HCCR optionally comprises one, two, or three conserved amino acid substitutions; and wherein the isolated antibody or antigen-binding portion thereof further comprises a light chain constant region (LCCR) comprising the amino acid sequence of SEQ ID NO: 8, wherein the LCCR optionally comprises one, two, or three conserved amino acid substitutions.

[0011] In some aspects, the VH of the antibody or antigen-binding portion further includes a heavy chain signal peptide (HCSP) at the N-terminus of the VH, said heavy chain signal peptide comprising the amino acids listed in SEQ ID NO: 15.

[0012] In another aspect, the antibody or antigen-binding portion thereof of this disclosure comprises VL, wherein the VL further comprises a light chain signal peptide (LCSP) at the N-terminus of the VL, the light chain signal peptide comprising the amino acids listed in SEQ ID NO: 16.

[0013] In one aspect, this disclosure provides an antibody or antigen-binding portion that is human-type or humanized from a different species, and may be IgG1, IgG2, IgG3 or IgG4.

[0014] In another aspect, this disclosure includes a pharmaceutical composition for treating, inhibiting, and / or suppressing solid tumors, comprising (i) an antibody or antigen-binding portion thereof disclosed herein; and (ii) a pharmaceutically acceptable carrier.

[0015] In another aspect, a polynucleotide molecule encoding the antibody or antigen-binding moiety disclosed herein has been considered and prepared, which, in one embodiment, may be an expression vector. In one embodiment, such an expression vector encodes a light chain signal peptide (LCSP) and / or a heavy chain signal peptide (HCSP), but does not contain introns. The expression vector can be used in host cells, such as Escherichia coli (E. coli).

[0016] In one aspect, this disclosure relates to a method for preventing, treating, and / or suppressing tumors in an individual, comprising administering an effective amount of an antibody or antigen-binding moiety to the individual. These tumors may include hepatocellular carcinoma, or solid tumors comprising melanoma and / or renal cell carcinoma (RCC).

[0017] In another aspect, this disclosure relates to a method for regulating the activity of one or more T cells in an individual in vitro, ex vivo, and / or in vivo, the method comprising: contacting T cells with an antibody or an antigen-binding portion thereof that preferentially binds to human programmed death receptor 1 (hPD-1), wherein the antibody blocks and / or regulates the binding of human PD-L1 and / or human PD-L2 to hPD-1, and wherein the antibody or the antigen-binding portion thereof comprises: a heavy chain variable region (VH) comprising SEQ ID NO: 5 or SEQ ID NO: 22; and a light chain variable region (VL) comprising SEQ ID NO: 6. In another aspect, the antibody or its antigen-binding portion may further comprise a heavy chain constant region (HCCR) comprising the amino acid sequence listed in SEQ ID NO: 7, and the HCCR optionally comprising one, two, or three conserved amino acid substitutions, and / or wherein the antibody or its antigen-binding portion further comprises a light chain constant region (LCCR) comprising the amino acid sequence of SEQ ID NO: 8, and wherein the LCCR optionally comprises one, two, or three conserved amino acid substitutions.

[0018] In another aspect, the methods disclosed herein can regulate T cells, such as one or more natural killer cells, one or more cytotoxic T cells, one or more helper T cells, one or more monocytes, one or more memory T cells and / or one or more regulatory T cells.

[0019] In one aspect, the antibody or antigen-binding moiety disclosed herein blocks human PD-L1 and human PD-L2 with human PD-1 at an IC50 of about 1 nM or lower.

[0020] Another aspect of this disclosure includes a method for producing an antibody or antigen-binding partial vector construct comprising culturing host cells containing an expression vector of interest under conditions that allow expression of the antibody or antigen-binding partial construct; and purifying and / or recovering the resulting antibody or antigen-binding partial vector construct from the culture.

[0021] In one aspect, the antibody or its antigen-binding portion for treating solid tumors may optionally contain another therapeutic agent. Such optional agents may be P1101 and / or another anti-human PD1 monoclonal antibody for treating solid tumors (including, but not limited to, RCC and / or melanoma).

[0022] This disclosure includes combinations for treating, inhibiting, preventing, and / or suppressing solid tumors in an individual, said combinations comprising an antibody or an antigen-binding portion thereof; and another therapeutic agent, said antibody or antigen-binding portion thereof comprising: a heavy chain variable region (VH) comprising SEQ ID NO: 5 or SEQ ID NO: 22; and a light chain variable region (VL) comprising SEQ ID NO: 6.

[0023] In addition, in the above combinations, the antibody or its antigen-binding portion further comprises a heavy chain constant region (HCCR), the heavy chain constant region comprising the amino acid sequence listed in SEQ ID NO: 7, and the HCCR optionally comprising one, two or three conserved modified amino acid substitutions; and wherein the antibody or its antigen-binding portion further comprises a light chain constant region (LCCR), the light chain constant region comprising the amino acid sequence of SEQ ID NO: 8, and wherein the LCCR optionally comprises one, two or three conserved modified amino acid substitutions.

[0024] In another aspect, this disclosure relates to a method for preventing, treating, and / or inhibiting solid tumors in an individual (e.g., a human), comprising administering effective amounts of P1101 and P1801 to said individual in a continuous or combined manner. The administration system can be in any continuous and / or sequential manner. In some aspects, P1101 is administered before or after P1801, and administration can be performed for various durations and combinations. The solid tumors treated include hepatocellular carcinoma, melanoma, and / or renal cell carcinoma.

[0025] In one embodiment, this disclosure provides an isolated antibody or antigen-binding portion thereof that preferentially binds to PD-1, comprising: a heavy chain complementarity-determining region 1 (VHCDR1) including the amino acid sequence listed in SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 21; a heavy chain complementarity-determining region 2 (VHCDR2) including the amino acid sequence listed in SEQ ID NO: 10; and / or a heavy chain complementarity-determining region 3 (VHCDR3) including the amino acid sequence listed in SEQ ID NO: 11, SEQ ID NO: 20 or SEQ ID NO: 23; and a light chain complementarity-determining region 1 (VLCDR1) including the amino acid sequence listed in SEQ ID NO: 12; a light chain complementarity-determining region 2 (VLCDR2) including the amino acid sequence listed in SEQ ID NO: 13; and / or a light chain complementarity-determining region 3 (VLCDR3) including the amino acid sequence listed in SEQ ID NO: 14.

[0026] In another embodiment, this disclosure provides a separable antibody or antigen-binding portion thereof that preferentially binds to PD-1, comprising one or more paratopes, said one or more paratopes comprising at least one of the following residues: Phe2, Ser5, Ser6 and Tyr7 of the heavy chain variable sequence (VHCDR1) of SEQ ID NO: 19.

[0027] At least one of the following residues: Phe2, Ser5, and Tyr6 of the heavy chain variable sequence (VHCDR1) of SEQ ID NO: 21; at least one of the following residues: Thr13, Gly14, Gly15, Gly16, Ser17, and Tyr18 of the heavy chain variable sequence (VHCDR2) of SEQ ID NO: 10; at least one of the following residues: Tyr33, Leu34, Asn35, Asp38, and Tyr39 of the heavy chain variable sequence (VHCDR3) of SEQ ID NO: 11; or at least one of the following residues: Ser31, Tyr33, Leu34, Asn35, Asp38, and Tyr39 of the heavy chain variable sequence (VHCDR3) of SEQ ID NO: 20.

[0028] In one embodiment, this disclosure provides a segregated antibody or antigen-binding portion thereof that preferentially binds to PD-1, comprising one or more complementary sites, said one or more complementary sites comprising: at least one of the following residues: Asn9 of the light chain variable sequence (VLCDR1) of SEQ ID NO: 12; at least one of the following residues: Tyr12 and Ser18 of the heavy chain variable sequence (VLCDR2) of SEQ ID NO: 13; or at least one of the following residues: Ser22, Asn23 and Trp25 of the heavy chain variable sequence (VLCDR3) of SEQ ID NO: 14.

[0029] Another embodiment of this disclosure provides a segregated antibody or its antigen-binding moiety that preferentially binds to PD-1, comprising one or more complementary sites, said one or more complementary sites comprising at least one of the following residues: Phe2, Ser5, Ser6 and Tyr7 of the heavy chain variable sequence (VHCDR1) of SEQ ID NO: 19; at least one of the following residues: Phe2, Ser5 and Tyr6 of the heavy chain variable sequence (VHCDR1) of SEQ ID NO: 21; at least one of the following residues: Thr13, Gly14, Gly15, Gly16, Ser17 and Tyr18 of the heavy chain variable sequence (VHCDR2) of SEQ ID NO: 10; at least one of the following residues: Tyr33, Leu34, Asn35, Asp38 and Tyr39 of the heavy chain variable sequence (VHCDR3) of SEQ ID NO: 11; at least one of the following residues: SEQ ID NO: Ser31, Tyr33, Leu34, Asn35, Asp38, and Tyr39 of the heavy chain variable sequence (VHCDR3) of SEQ ID NO: 20; at least one of the following residues: Asn9 of the light chain variable sequence (VLCDR1) of SEQ ID NO: 12; at least one of the following residues: Tyr12 and Ser18 of the heavy chain variable sequence (VLCDR2) of SEQ ID NO: 13; or at least one of the following residues: Ser22, Asn23, and Trp25 of the heavy chain variable sequence (VLCDR3) of SEQ ID NO: 14.

[0030] In one embodiment, this disclosure provides an isolated antibody or antigen-binding portion thereof that preferentially binds to PD-1, comprising: a heavy chain variable region (VH) comprising SEQ ID NO: 9 and a light chain variable region (VL) comprising SEQ ID NO: 6; or a heavy chain variable region (VH) comprising SEQ ID NO: 18 and a light chain variable region (VL) comprising SEQ ID NO: 6; or a heavy chain variable region (VH) comprising SEQ ID NO: 19 and a light chain variable region (VL) comprising SEQ ID NO: 6; or a heavy chain variable region (VH) comprising SEQ ID NO: 21 and a light chain variable region (VL) comprising SEQ ID NO: 6.

[0031] In one embodiment, this disclosure provides an isolated antibody or antigen-binding portion thereof that preferentially binds to PD-1, comprising: a heavy chain variable region (VH) comprising SEQ ID NO: 5 or a heavy chain variable region (VH) comprising SEQ ID NO: 22; and a light chain variable region (VL) comprising SEQ ID NO: 6.

[0032] In any of the embodiments including an antibody or an antigen-binding portion thereof, the antibody or antigen-binding portion thereof may further include a heavy chain constant region (HCCR) comprising the amino acid sequence listed in SEQ ID NO: 7; and wherein the HCCR optionally comprises one, two or three conserved modified amino acid substitutions.

[0033] In any of the embodiments including an antibody or an antigen-binding portion thereof, the antibody or antigen-binding portion thereof may further include a light chain constant region (LCCR) comprising the amino acid sequence of SEQ ID NO: 8; and wherein the LCCR optionally comprises one, two or three conserved modified amino acid substitutions.

[0034] In any of the embodiments including an antibody or an antigen-binding portion thereof, the antibody or antigen-binding portion thereof may further comprise: a heavy chain constant region (HCCR) comprising the amino acid sequence listed in SEQ ID NO: 7, wherein the HCCR optionally comprises one, two, or three conserved amino acid substitutions; and a light chain constant region (LCCR) comprising the amino acid sequence of SEQ ID NO: 8, wherein the LCCR optionally comprises one, two, or three conserved amino acid substitutions.

[0035] In any of the embodiments including an antibody or its antigen-binding portion, the VH may further include a heavy chain signal peptide (HCSP) at the N-terminus of the VH, the heavy chain signal peptide comprising the amino acids listed in SEQ ID NO: 15. In any of the embodiments including an antibody or its antigen-binding portion, the VL may further include a light chain signal peptide (LCSP) at the N-terminus of the VL, the light chain signal peptide comprising the amino acids listed in SEQ ID NO: 16. In any of the embodiments including an antibody or its antigen-binding portion, the antibody may be a human or humanized antibody. In any of the embodiments including an antibody or its antigen-binding portion, the antibody may be an IgG4 isotype.

[0036] In one aspect, this disclosure provides a pharmaceutical composition for treating, inhibiting, and / or suppressing solid tumors, comprising (i) an antibody or an antigen-binding portion thereof disclosed in any of the examples listed herein; and (ii) a pharmaceutically acceptable carrier.

[0037] In one embodiment, this disclosure provides a polynucleotide molecule that encodes an antibody or antigen-binding portion of any of the embodiments listed herein.

[0038] In one embodiment, this disclosure provides an expression vector comprising a polynucleotide molecule encoding an antibody or antigen-binding moiety disclosed in any of the embodiments listed herein, wherein the expression vector encoding LCSP and / or HCSP does not contain introns. In one embodiment, this disclosure provides a host cell comprising the expression vector.

[0039] In one embodiment, this disclosure provides a method for preventing, treating, and / or suppressing tumors in an individual, comprising the step of administering to the individual an effective amount of an antibody or antigen-binding portion disclosed in any of the embodiments listed herein. The tumor includes hepatocellular carcinoma, or a solid tumor comprising melanoma and / or renal cell carcinoma (RCC).

[0040] In one embodiment, this disclosure provides a method for regulating the activity of one or more T cells in an individual in vitro, ex vivo, and / or in vivo, the method comprising the steps of: contacting the T cells with an antibody or its antigen-binding portion that preferentially binds to human programmed death receptor 1 (hPD-1), wherein the antibody blocks and / or regulates the binding of human PD-L1 and / or human PD-L2 to hPD-1, and wherein the antibody or its antigen-binding portion comprises: a heavy chain complementarity-determining region 1 (VHCDR1) comprising the amino acid sequence listed in SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 21; a heavy chain complementarity-determining region 2 (VHCDR2) comprising the amino acid sequence listed in SEQ ID NO: 10; and / or a heavy chain complementarity-determining region 3 (VHCDR3) comprising the amino acid sequence listed in SEQ ID NO: 11, SEQ ID NO: 20, or SEQ ID NO: 23; and a light chain complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence listed in SEQ ID NO: 12 ...0; and a light chain complementarity-determining region 2 (VLCDR1) comprising the amino acid sequence listed in SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 21; and a light chain complementarity-determining region 3 (VHCDR3) comprising the amino acid sequence listed in SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO: 19, or The light chain complementarity-determining region 2 (VLCDR2) of the amino acid sequence listed in SEQ ID NO: 13, and / or the light chain complementarity-determining region 3 (VLCDR3) of the amino acid sequence listed in SEQ ID NO: 14.

[0041] In one embodiment of a method for modulating the activity of one or more T cells in an individual in vitro, ex vivo, and / or in vivo, the antibody or its antigen-binding portion further comprises a heavy chain constant region (HCCR) comprising the amino acid sequence listed in SEQ ID NO: 7, and the HCCR optionally comprises one, two, or three conserved amino acid substitutions. In the method, the antibody or its antigen-binding portion may further comprise a light chain constant region (LCCR) comprising the amino acid sequence of SEQ ID NO: 8, wherein the LCCR optionally comprises one, two, or three conserved amino acid substitutions. In the method, the one or more T cells may comprise one or more natural killer cells, one or more cytotoxic T cells, one or more helper T cells, one or more monocytes, one or more memory T cells, and / or one or more regulatory T cells. In the method, the antibody or antigen-binding portion may have an IC50 concentration of about 1 nM or lower. 50 Blocking the binding of human PD-L1 and human PD-L2 to human PD-1.

[0042] In one aspect, this disclosure provides a method for generating an antibody or antigen-binding partial vector construct comprising culturing host cells disclosed in any of the examples listed herein under conditions that allow expression of the antibody or antigen-binding partial construct; and purifying and / or recovering the resulting antibody or antigen-binding partial vector construct from the culture.

[0043] In another aspect, this disclosure provides an antibody or antigen-binding portion thereof for treating solid tumors, comprising: a heavy chain variable region (VH) comprising SEQ ID NO: 5 or a heavy chain variable region (VH) comprising SEQ ID NO: 22; a light chain variable region (VL) comprising SEQ ID NO: 6; and optionally comprising another therapeutic agent. The antibody or antigen-binding portion thereof may further comprise: a heavy chain constant region (HCCR) comprising the amino acid sequence listed in SEQ ID NO: 7, wherein the HCCR optionally comprises one, two, or three conserved modified amino acid substitutions; and a light chain constant region (LCCR) comprising the amino acid sequence of SEQ ID NO: 8, wherein the LCCR optionally comprises one, two, or three conserved modified amino acid substitutions; and optionally comprising another therapeutic agent. The other therapeutic agent may comprise P1101, P1801, and / or another anti-human PD1 monoclonal antibody. In the antibody or antigen-binding portion thereof for use, the solid tumor may comprise hepatocellular carcinoma, renal cell carcinoma, and / or melanoma.

[0044] In one embodiment, this disclosure provides a combination for treating, inhibiting, preventing, and / or suppressing solid tumors in an individual, wherein the combination comprises an antibody or an antigen-binding portion thereof; and another therapeutic agent, wherein the antibody or its antigen-binding portion comprises: a heavy chain variable region (VH) comprising the heavy chain variable region (VH) of SEQ ID NO: 5 or the heavy chain variable region (VH) comprising the heavy chain variable region (VH) comprising the heavy chain variable region (VL) comprising the light ... In the combination, P1101, P1801, and / or another anti-human PD1 monoclonal antibody may be administered sequentially. In the combination, P1101 and / or P1801 are administered prior to another anti-human PD1 monoclonal antibody. In the combination, the solid tumor may include hepatocellular carcinoma, melanoma, and / or renal cell carcinoma.

[0045] In one embodiment, this disclosure provides a recombinant protein comprising (i) an antibody or antigen-binding moiety disclosed herein; and (ii) an optional tag sequence for expression for detection and / or purification.

[0046] In another embodiment, any of the embodiments including an antibody or its antigen-binding portion may be used in a method of treating cancer, wherein the method comprises administering an isolated antibody or its antigen-binding portion to an individual. In one embodiment, the cancer includes hepatocellular carcinoma, melanoma, and / or renal cell carcinoma.

[0047] In one embodiment, any of the embodiments including an antibody or its antigen-binding portion can be used to manufacture an agent for therapeutic and / or preventative treatment of cancer. In one embodiment, the cancer includes hepatocellular carcinoma, melanoma, and / or renal cell carcinoma. Attached Figure Description

[0048] To gain a more complete understanding of the features and advantages of this disclosure, reference is now made to the detailed description of this disclosure and the accompanying drawings, wherein:

[0049] Figures 1(a) to 1(r)The amino acid sequences of P1801 are shown in Figure 1(a). Figure 1(a) shows the complete heavy chain of P1801, including three CDRs, a variable domain, and a constant region. It also includes a heavy chain signal peptide that will eventually be cleaved by the host cell. The complete heavy chain sequence is represented as SEQ ID NO: 1. Figure 1(b) shows the complete light chain of P1801, including three CDRs, a variable domain, and a constant region. It also includes a light chain signal peptide that will eventually be cleaved by the host cell. The complete light chain sequence is represented as SEQ ID NO: 2. Figure 1(c) shows the heavy chain of P1801 without the heavy chain signal peptide (SEQ ID NO: 3). Figure 1(d) shows the light chain of P1801 without the light chain signal peptide (SEQ ID NO: 4). Figure 1(f) shows the complete variable region of the heavy chain of P1801 (SEQ ID NO: 5). Figure 1(g) shows the complete variable region of the light chain of P1801 (SEQ ID NO: 6). Figure 1(h) shows the heavy chain constant region of P1801 (SEQ ID NO: 7). Figure 1(i) shows the light chain constant region of P1801 (SEQ ID NO: 8). Figure 1(j) shows the heavy chain CDR1 of P1801 (SEQ ID NO: 9). Figure 1(k) shows the heavy chain CDR2 of P1801 (SEQ ID NO: 10). Figure 1(l) shows the heavy chain CDR3 of P1801 (SEQ ID NO: 11). Figure 1(m) shows the light chain CDR1 of P1801 (SEQ ID NO: 12). Figure 1(n) shows the light chain CDR2 of P1801 (SEQ ID NO: 13). Figure 1(o) shows the light chain CDR3 of P1801 (SEQ ID NO: 14). Figure 1(p) shows the heavy chain signal peptide of P1801 (SEQ ID NO: 15). Figure 1(q) shows the light chain signal peptide of P1801 (SEQ ID NO: 16). Figure 1(r) shows the hinge sequence located in the heavy chain (SEQ ID NO: 17). Figure 1(s) shows the alternative heavy chain CDR1 of P1801 (SEQ ID NO: 18). Figure 1(t) shows the alternative heavy chain CDR1 of P1801 (SEQ ID NO: 19). Figure 1(u) shows the alternative heavy chain CDR1 of P1801 (SEQ ID NO: 20). Figure 1(v) shows the alternative heavy chain CDR1 of P1801 (SEQ ID NO: 21). Figure 1(w) shows the alternative intact heavy chain variable region of P1801 (SEQ ID NO: 22). Figure 1(x) shows the alternative heavy chain CDR3 of P1801 (SEQ ID NO: 23).

[0050] Figure 2This indicates the chemical structure of P1101. IFN represents human interferon α2b. mPEG represents a polyethylene glycol polymer, and each mPEG has a molecular weight of approximately 10 kD to 30 kD, and / or each mPEG has a molecular weight of approximately 20 kD.

[0051] Figure 3 This represents a competitive ELISA used to test the interference resistance of selected mice against hPD-1 mAb (mP1801) compared to commercially known anti-PD1 mAb.

[0052] Figures 4(a) to 4(c) Different embodiments of administration regimens of combinations of molecules of this disclosure (e.g., P1101 and an anti-PD1 monoclonal antibody, such as P1801) are shown. Solid and dashed arrows may represent molecule 1, such as P1101; and molecule 2, such as P1801, an anti-PD1 monoclonal antibody, or vice versa.

[0053] Figure 5 This illustrates another different embodiment of the administration regimen of the combination of molecules of this disclosure. For example, molecule 1 may be P1101 or an anti-PD1 monoclonal antibody, such as P1801. Molecule 2 may be P1101 or an anti-PD1 monoclonal antibody, such as P1801, or vice versa.

[0054] Figure 6 This represents yet another different embodiment of the administration regimen of the combination of molecules disclosed in this disclosure. For example, molecule 1 may be P1101 or an anti-PD1 monoclonal antibody, such as P1801. Molecule 2 may be P1101 or an anti-PD1 monoclonal antibody, such as P1801.

[0055] Figure 7 illustrates another embodiment of the administration regimen of the combination of molecules disclosed in this disclosure. For example, Figure 7(a) shows that molecule 1 can be hIgG4 (as an isotope control), P1101, or an anti-PD1 monoclonal antibody, such as P1801, cimiplimab, pembrolizumab, or novolumab administered alone. Figure 7(b) shows an alternation between molecules 1 and 2, wherein molecule 1 can be mP1101, molecule 2 can be P1801, and vice versa.

[0056] Figure 8(a) and 8(b) The binding assays of P1801 against various commercially available anti-PD1 mAbs against the PD1 antigen are shown. The Y-axis represents wavelength, and the x-axis represents time in seconds. As shown in Figure 8(a), the second line from the top represents P1801 and is marked with an arrow. As shown in Figure 8(b), the third line from the top represents P1801 and is marked with an arrow.

[0057] Figure 9 This indicates the binding specificity of P1801. P1801 specifically binds to PD-1, but not to PD-L1, PD-L2, CTLA4, or CD28 because these are flat and undetectable.

[0058] Figure 10 This represents the average RCC tumor volume from the seven different groups described in the protocol of Example 9.

[0059] Figure 11 This represents the actual RCC tumor measurement from Group 1 of Example 9.

[0060] Figure 12 This represents the actual RCC tumor measurement from group 6 of Example 9.

[0061] Figure 13 This indicates five different groups used to determine the effective dose of P1101 + P1801 in individuals.

[0062] Figure 14 This indicates the effective dose relationship between P1101 + P1801. Figure 13 Further optimization of dose groups. The top ring is used for RCC, and the bottom ring is used for melanoma.

[0063] Figure 15 For another instance of another group.

[0064] Figure 16 Images generated for the final 3D reconstruction of PD1-AcroBio + PS 00066 + PS00067 using ChimeraX at a nominal resolution of 2.9A.

[0065] Figure 17 The final 2.9A map image generated using ChimeraX is colored using local resolution values ​​calculated by cryoSPARC 4.4.

[0066] Figure 18 The image shows the structure of the P1801 / PD1 complex as resolved by cryo-electron microscopy.

[0067] Figure 19 The image shows the structure of the P1801 / PL1 / PD1 complex as resolved by cryo-electron microscopy, revealing the overlap between P1801 / PD1 and PD-L1 / PD1.

[0068] Figure 20 Image for locating the P1801 epitope.

[0069] Figure 21Image for PD-L1 epitope localization.

[0070] Figure 22 The model of P1801 CDR at the PD-1 interface.

[0071] Figure 23 This is a graph showing the binding of epitopes to anti-PD-1 antibodies.

[0072] Although various embodiments of this disclosure are described in detail below, and their use is permitted, it should be understood that this disclosure provides numerous applicable inventive concepts that can be practiced in a wide variety of specific situations. The specific embodiments discussed herein are merely illustrative of particular ways of carrying out and using this disclosure and do not limit the scope of this disclosure.

[0073] To aid in understanding this disclosure, several terms are defined below. The terms defined herein have meanings as commonly understood by one of ordinary skill in the art related to this disclosure. For example, the terms “a / an” and “the” are not intended to refer only to a singular entity, but rather to include general categories that can be used to describe a particular instance. The terms used herein are used to describe specific embodiments of this disclosure, but their use does not limit this disclosure beyond what is set forth in the claims.

[0074] i. Definition

[0075] As used herein, the term "isolated molecule" refers to a molecule (where the molecule is, for example, a polypeptide, polynucleotide, or antibody) that, due to its origin or derivative, (1) does not associate with the naturally associated components that accompany it in its native state; (2) is substantially free of other molecules from the same source (e.g., species, cells expressing it, libraries, etc.); (3) is expressed by cells from a different species; or (4) is not found in nature. Therefore, a chemically synthesized molecule or expressed in a cellular system different from the system of its natural origin will be "isolated" from its naturally associated components. Isolation can also render a molecule substantially free of its naturally associated components using purification techniques well-known in the art. Molecular purity or homogeneity can be determined by a variety of methods known in the art. For example, the purity of a polypeptide sample can be determined using polyacrylamide gel electrophoresis and gel staining to visualize the polypeptide using techniques well-known in the art. For certain purposes, higher resolution can be provided by using HPLC or other well-known methods for purification in the art.

[0076] "Antibody" is an immunoglobulin molecule capable of specifically binding to targets such as carbohydrates, polynucleotides, lipids, and peptides via at least one antigen recognition site located in a variable region of an immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also, unless otherwise specified, any antigen-binding portion of the intact antibody that competes for specific binding, and any other modified configuration of an immunoglobulin molecule containing an antigen recognition site. Antigen-binding portions include, for example, Fab, Fab', F(ab')2, Fd, Fv, domain antibodies (dAb, such as shark and camel antibodies), fragments including complementarity-determining regions (CDRs), single-chain variable fragment antibodies (scFv), macrobody, minibody, intrabody, diabody, triabody, tetrabody, v-NAR, and bis-scFv, as well as peptides containing at least a portion of an immunoglobulin sufficient to bind a specific antigen to the peptide. Antibodies include any class of antibodies, such as IgG, IgA, or IgM (or their subclasses), and antibodies do not necessarily belong to any specific class. Immunoglobulins can be classified into different classes based on the antibody amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are known in their respective fields.

[0077] The "variable region" of an antibody refers to the antibody light chain variable region or antibody heavy chain variable region, either alone or in combination. As is known in the art, both the heavy chain and light chain variable regions consist of four frame regions (FRs), which are linked by three complementarity-determining regions (CDRs), also known as hypervariable regions. The heavy and light chain variable regions facilitate the formation of the antibody's antigen-binding site. If a variant of the target variable region is desired, particularly a variant with substitutions in amino acid residues outside the CDR regions (i.e., within the frame regions), appropriate amino acid substitutions, preferably conserved substitutions, can be identified by comparing the target variable region with variable regions of other antibodies containing CDR1 and CDR2 sequences belonging to the same classical class as the target variable region.

[0078] In some embodiments, the CDR is clearly delineated and the residues containing the antibody binding site are identified by resolving the structure of the antibody and / or the structure of the antibody-ligand complex. In some embodiments, this can be achieved by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In some embodiments, various analytical methods can be used to identify or estimate the CDR region. Examples of these methods include, but are not limited to, Kabat definition, Chothia definition, AbM definition, contact definition, and configuration definition. In one embodiment, the CDR of this disclosure is determined by the Kabat definition. In another embodiment, the CDR is determined by the Chothia definition.

[0079] As used in this article, the “EU index” or “EU numbering” system is based on the sequence number of the first human IgG (EU antibody) sequenced. Because the most commonly used reference for this convention is the Kabat sequence manual, the EU index is sometimes mistakenly confused with the Kabat index. The EU index does not provide for insertions and deletions, so in some cases, comparisons of IgG positions between IgG subclasses and species may be ambiguous, especially in hinge regions. Nevertheless, this convention is sufficient for simple comparisons of Fc regions in many Fc structure-function studies.

[0080] As used in this article, the “configurational definition” of a CDR refers to the position of a CDR that can be identified as a residue that contributes enthalpy to antigen binding.

[0081] As used herein, CDR may refer to any CDR defined by any method known in the art, including combinations of methods. The methods used herein may utilize CDRs defined according to any of these methods. For any given embodiment containing more than one CDR, a CDR may be defined according to any of the following: Kabat definition (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), IMGT definition, Chothia definition (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)), Martin definition, extended definition, AbM definition (using Oxford Molecular's AbM antibody model software), contact definition, conformation definition, and / or any CDR determination method known in the art.

[0082] CDRs are generally considered to be structured loops that participate in antigen binding and exhibit a highly variable amino acid composition. Different numbering schemes or definitions of CDR length can be used to define CDRs based on the antibody amino acid sequence. For example, the Kabat (and IMGT) CDR definition is based on sequence alignment, while the Chothia CDR definition better reflects the loop structure in the antibody's 3D architecture. The Kabat numbering scheme is generally considered the widely adopted standard for antibody residue numbering. The Chothia numbering scheme is based on antibody structure alignment and ensures a better correspondence with structural loops. The Chothia numbering scheme is characterized by topologically aligned residues from different antibodies being numbered at the same positions, and the Chothia CDR definition corresponding to structural antigen-binding loops in most antibody sequences. CDRs defined on highly variable amino acids according to the Kabat and Chothia nomenclature may have some positional shifts and / or deviations in loop length.

[0083] The Martin numbering scheme focuses on the structural alignment of different frame regions of unconventional length. It highlights residues absent in most sequences and structures, defining these residues as deletion sites and suggesting corrections for insertion points within frame region 3 of the heavy chain domain. The Martin numbering scheme uses the numbering software ABnum and provides a numbering scheme comprised of the Chothia numbering system corrected by ABnum software, which integrates sequences from the Kabat, IMGT, and PDB databases. The IMGT numbering scheme is a reference in immunogenetics and immunoinformatics, providing a standardized numbering system for all protein sequences in the immunoglobulin superfamily, including variable domains from antibody light and heavy chains and T-cell receptor chains from different species. The IMGT numbering scheme is based on germline V gene amino acid sequence alignment. The IMGT numbering scheme covers all variable domains and various tools have been developed for analyzing full-length sequences. Alternatively, any other novel method not mentioned in this disclosure may be used to determine CDRs.

[0084] As is known in the field, the “constant region” of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.

[0085] As used herein, a “monoclonal antibody” refers to an antibody obtained from a largely homogeneous population of antibodies, meaning that the individual antibodies constituting the population are identical except for the possible small number of naturally occurring mutations. Monoclonal antibodies are highly specific for a single antigenic site. Furthermore, in contrast to polyclonal antibody formulations, which typically consist of different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. The modifier “monoclonal” indicates that the antibody is obtained from a largely homogeneous population of antibodies and should not be construed as requiring any special method to produce the antibody. For example, monoclonal antibodies used according to this disclosure can be prepared by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or by the recombinant DNA method described, for example, in U.S. Patent No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries produced using techniques known in the art.

[0086] As used herein, a “humanized” antibody refers to a form of non-human (e.g., murine) antibody that is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequence of the antibody) containing a minimal sequence derived from a non-human immunoglobulin. A humanized antibody is a human immunoglobulin (recipient antibody) in which residues of a recipient’s CDR are replaced by residues of a CDR from a non-human species (donor antibody) having the desired specificity, affinity, and ability. Humanized antibodies may contain residues that are not present in the recipient antibody or in the introduced CDR or frame sequence, but such residues are included to further improve and optimize antibody performance.

[0087] Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” all refer to both of the following: (1) a treatment measure that cures, alleviates, or reduces the symptoms of a diagnosed pathological condition or disease and / or prevents its progression, and / or (2) a preventive or preventative measure that prevents and / or slows the development of a targeted pathological condition or disease. Therefore, those who need treatment include: people who already have the disease; people who are susceptible to the disease; and people who need to prevent the disease. In some respects, according to the methods of this disclosure, if a patient shows, for example, overall, partial, or transient remission of a certain type of cancer, then the individual's cancer is successfully “treated”.

[0088] "Human antibody" is an antibody whose amino acid sequence corresponds to that of human-derived antibodies and / or is prepared using any of the human antibody preparation techniques disclosed herein. This definition of human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues.

[0089] The term "chimeric antibody" refers to an antibody whose variable region sequence originates from one species and whose constant region sequence originates from another species, such as an antibody whose variable region sequence originates from a mouse antibody and whose constant region sequence originates from a human antibody.

[0090] As used herein, the terms “inhibition” and / or “modulation” of PDL1 and / or PDL2 activity refer to the ability of an anti-PD1 binding molecule (e.g., an antibody or its antigen-binding fragment) to bind to PD1, and in some aspects, to inhibit PDL1 and / or PDL2 activity by reducing it by at least about 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99%, or 100%.

[0091] The term "epitope" refers to a portion of a molecule that can be recognized by an antibody and bind to one or more of its antigen-binding regions. Epitopes typically consist of surface clusters of molecules such as amino acids or sugar side chains and possess specific three-dimensional structural characteristics and specific charge properties. In some embodiments, an epitope may be a protein epitope. Protein epitopes can be linear or conformational. In a linear epitope, all interaction sites between the protein and the interacting molecule (e.g., an antibody) are linear along the primary amino acid sequence of the protein. A "nonlinear epitope" or "conformational epitope" comprises a discontinuous polypeptide (or amino acid) within an antigen protein to which an antibody specific to the epitope binds.

[0092] As used herein, the term "antigenic epitope" is defined as a portion of an antigen that can specifically bind to an antibody, and can be determined by any method well-known in the field (e.g., by routine immunoassay). Once the desired epitope on the antigen is identified, an antibody against that epitope can be generated. Alternatively, during the exploration process, the generation and characterization of antibodies can elucidate information about the desired epitope. Antibodies that bind to the same epitope can be competitively screened. This is achieved by conducting competitive and cross-competitive studies to find antibodies that compete or cross-competitively bind to PD-1, for example, antibodies that compete to bind to the antigen.

[0093] This antibody or its antigen-binding fragment contains "complementary" and "non-complementary" amino acid residues. The "complementary" amino acid of this antibody or its antigen-binding fragment may have a nucleus, for example, within approximately 4 Å of the nucleus of an antibody or its antigen-binding moiety that preferentially binds to PD-1 isoforms. Since each isoform forms a structurally different complex with this antibody or its antigen-binding fragment, the complementary residues for each isoform may be different. An "antibody or its antigen-binding moiety that preferentially binds to PD-1 complementary residues" may, for example, have a nucleus within approximately 4 Å of the nucleus of a human antibody or its antigen-binding moiety that preferentially binds to PD-1. An "antibody or its antigen-binding moiety that preferentially binds to PD-1 complementary residues" may have a nucleus, for example, within approximately 4 Å of the nucleus of all isoforms.

[0094] Regardless of their position within the CDR or FW (framework) region, residues can be named complementary residues, defined using the Kabat nomenclature. For example, many complementary residues are located within the CDR region, as shown in Table 15. However, some complementary residues are located within the FW region. A "non-complementary" amino acid is any amino acid in the antibody or its antigen-binding fragment that is not a "complementary" amino acid, regardless of whether the residue is located in the CDR or FW region.

[0095] The terms “programmed cell death 1,” “programmed cell death 1,” “protein PD-1,” “PD-1,” “PD1,” “PDCD1,” “hPD-1,” and “hPD-I” are all used interchangeably and include variants, isoforms, species homologs of human PD-1, and analogs that share at least one epitope with PD-1. The complete PD-1 sequence is available at GenBank accession number U64863 and is incorporated herein by reference.

[0096] The term "agonist" refers to a substance that promotes (i.e., induces, induces, enhances, or increases) the biological activity or action of another molecule. The term agonist encompasses substances that bind to receptors (e.g., antibodies) and substances that promote receptor function without binding to them (e.g., by activating related proteins).

[0097] The term "antagonist" or "inhibitor" refers to a substance that prevents, blocks, inhibits, neutralizes, or reduces the biological activity or action of another molecule (such as a receptor).

[0098] As used herein, "anti-PD-1 antibody" refers to an antibody capable of inhibiting PD-1 biological activity and / or one or more downstream events mediated by PD-1. Anti-PD-1 antibody encompasses antibodies that block, antagonize, inhibit, or reduce (to any extent, including significantly) PD-1 biological activity, including PD-1-mediated downstream events such as PD-L1 binding and downstream signaling, PD-L2 binding and downstream signaling, T cell proliferation inhibition, T cell activation inhibition, IFN secretion inhibition, IL-2 secretion inhibition, TNF secretion inhibition, IL-10 induction, and suppression of anti-tumor immune responses. For the purposes of this disclosure, it should be understood that the term "anti-PD-1 antibody" (interchangeably referred to as "PD-1 antibody") encompasses all previously identified terms, names, and functional states and properties that substantially invalidate, reduce, or neutralize PD-1 itself, PD-1 biological activity, or the results of such biological activity to any extent. In some embodiments, anti-PD-1 antibodies bind to PD-1 and upregulate anti-tumor immune responses. This document provides examples of anti-PD-1 antibodies, such as P1801. In one embodiment, P1801 is first produced in a mouse form and subsequently humanized for human use. In some embodiments, known forms of mouse anti-PD-1 monoclonal antibodies, RMP 1-14, can be used to demonstrate efficacy in mouse models.

[0099] The terms “polypeptide,” “oligopeptide,” “peptide,” and “protein” are used interchangeably herein to refer to amino acid chains of any length. The chain may be linear or branched and may contain modified amino acids and / or interspersed with non-amino acids. The term also covers amino acid chains that have been modified naturally or through intervention; said intervention is, for example, the formation of disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation, or any other operation or modification, such as binding to a labeled component. This definition also includes, for example, one or more analogues containing amino acids (including, for example, non-natural amino acids) and other modified polypeptides known in the art. It should be understood that polypeptides may exist as single chains or related chains.

[0100] As is known in the art, the terms “polynucleotide” or “nucleic acid” as used interchangeably herein refer to a nucleotide chain of any length and include both DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerases. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and their analogs. If modifications are present, the nucleotide structure may be modified before or after chain assembly. The nucleotide sequence may contain interspersed non-nucleotide components. Polynucleotides may be further modified after polymerization, for example, by binding to labeled components. Other types of modifications include, for example, "caps"; substitution of one or more naturally occurring nucleotides with analogs; internucleotide modifications, such as those with non-electrolyte bonds (e.g., methyl phosphonate, triphosphate, aminophosphate, carbamate, etc.) and those with charged bonds (e.g., thiophosphate, dithiophosphate, etc.); modifications containing side groups such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); modifications containing intercalating agents (e.g., acridine, psoralen, etc.); modifications containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.); modifications containing alkylating agents; modifications with modified bonds (e.g., α-mutated isomers of nucleic acids); and unmodified forms of polynucleotides. Furthermore, any hydroxyl group generally present in sugars can be, for example, replaced by a phosphonate group or a phosphate group, protected by a standard protecting group, or activated to prepare for forming additional bonds with other nucleotides, or can be bound to a solid carrier. The 5' and 3' OH groups may be phosphorylated or partially substituted by an amine or an organic end-capping group of 1 to 20 carbon atoms. Other hydroxyl groups may also be derived as standard protecting groups. Polynucleotides may also contain similar forms of ribose or deoxyribose known in the art, including, for example, 2'-O-methylribose, 2'-O-allylribose, 2'-fluororibose or 2'-azidoribose, carbocyclic sugar analogs, α-mutameric or β-mutameric sugars, epimeric sugars (e.g., arabinose, xylose or lyxose); pyranose, furanose, sedoheptulose, acyclic analogs and non-basic nucleoside analogs (e.g., methylriboside). One or more phosphodiester bonds may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate ester group is replaced by P(O)S (“thioester group”), P(S)S (“dithioester group”), (O)NR2 (“amide ester group”), P(O)R, P(O)OR', CO or CH2 (“methyl acetal”), wherein each R or R' is independently H or a substituted or unsubstituted alkyl (1 to 20 C), aryl, alkenyl, cycloalkyl, cycloalkenyl or aromatic aldehyde group optionally containing an ether (-O-) bond.Not all bonds in a polynucleotide need to be identical. The foregoing description applies to all polynucleotides mentioned in this article, including RNA and DNA.

[0101] Antibodies that “preferentially bind” or “specifically bind” (used interchangeably herein) to epitopes are terms understood in the art, and methods for determining such specificity or preferential binding are also known in the art. A molecule is said to exhibit “specific binding” or “preferential binding” if its reaction or association with a particular cell or substance is more frequent, faster, longer-lasting, and / or has a greater affinity than with alternative cells or substances. An antibody is “specifically bound” or “preferentially bound” to a target if its binding to a target has a greater affinity, is easier, and / or lasts longer than its binding to other substances. For example, an antibody that specifically or preferentially binds to a PD-1 epitope is an antibody that binds to that epitope with a greater affinity, is easier, and / or lasts longer than it binds to other PD-1 epitopes or non-PD-1 epitopes. It should also be understood from this definition that, for example, an antibody (or part or epitope) that specifically or preferentially binds to a first target may specifically or preferentially bind to a second target, or may not specifically or preferentially bind to a second target. Therefore, "specific binding" or "preferred binding" does not necessarily require (although it may include) exclusive binding. Generally speaking, but not necessarily, when binding is mentioned, it refers to preferred binding.

[0102] As used in this article, “generally pure” means material that is at least 50% pure (i.e., free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0103] "Host cell" includes individual cells or cell cultures that may be, or have been, a recipient of a vector for incorporating polynucleotide intercalations. Host cells include progeny of a single host cell, and progeny may not necessarily be completely identical to the original parent cell (in terms of morphology or genomic DNA complement) due to natural, accidental, or intentional mutations. Host cells include cells in vivo transfected with one or more polynucleotides disclosed herein. An example is *Escherichia coli*.

[0104] Unless otherwise indicated, as used herein, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the range stated, and, where appropriate, to include fractions (e.g., one-tenth and one-hundredth of an integer).

[0105] As used in this field, "Fc receptor" and "FcR" describe receptors that bind to the Fc region of an antibody. Preferably, the FcR is a naturally occurring human FcR. Furthermore, the FcR is preferably an FcR (γ receptor) that binds to IgG antibodies and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternating splice forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), both with similar amino acid sequences, differing primarily in their cytoplasmic domains. FcRs are those reviewed and known in this field. "FcR" also includes the neonatal receptor FcRn, responsible for transferring maternal IgG to the fetus.

[0106] As used herein with respect to antibodies, the term "competition" means that the binding of a first antibody or its antigen-binding moiety to an epitope is sufficiently similar to the binding of a second antibody or its antigen-binding moiety to such that the binding of the first antibody to its homologous epitope in the presence of the second antibody is detectably reduced compared to the binding of the first antibody in the absence of the second antibody. Alternatively, the binding of the second antibody to its epitope may also be detectably reduced in the presence of the first antibody, but this is not always the case. That is, the first antibody may inhibit the binding of the second antibody to its epitope, while the second antibody may not inhibit the binding of the first antibody to its respective epitope. However, when each antibody detectably inhibits the binding of another antibody to its homologous epitope or ligand, whether the degree of inhibition is the same, greater, or less, these antibodies are said to "cross-compete" for binding to their respective epitopes. This disclosure covers both competitive and cross-competitive antibodies. Regardless of the mechanism by which this competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or a portion thereof), those skilled in the art will understand, based on the teachings provided herein, that such competitive and / or cross-competitive antibodies are applicable to the methods disclosed herein.

[0107] A “functional Fc region” possesses at least one effector function of the native Fc region. Exemplary effector functions include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity; phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptors). These effector functions typically require a combination of the Fc region and a binding domain (e.g., antibody variable domain), and can be assessed using various assays known in the field.

[0108] The “natural sequence Fc region” comprises an amino acid sequence identical to that of an Fc region found in nature. The “variant Fc region” comprises an amino acid sequence that differs from the natural sequence Fc region due to at least one amino acid modification, but retains at least one effector function of the natural sequence Fc region. Preferably, compared to the natural sequence Fc region or the Fc region of the parent peptide, the variant Fc region has at least one amino acid substitution in the natural sequence Fc region or the Fc region of the parent peptide, for example, about one to about ten amino acid substitutions, or about one to about five amino acid substitutions. The variant Fc region described herein has at least about 80% sequence identity with the natural sequence Fc region and / or the Fc region of the parent peptide, at least about 90% sequence identity with it, and at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least 99.5% sequence identity with it.

[0109] As used herein, the term "dose" refers to the amount of one or more active ingredients intended to be administered to an individual each time. The term "dosage" is sometimes referred to as the plural form of the term dose. Each dose and dosage may contain one or more active ingredients that are constant and / or variable, and the amount of active ingredient in each dose or dosage may vary, as appropriate. A unit dose or dosage may be in the form of tablets, capsules, pouches, or liquids. For example, a dose (e.g., in the form of one or two separate tablets) may be 0.5 mg of compound A, or may include 0.5 mg of compound A and 0.1 mg of compound B. As appropriate, dose and dosage may be used interchangeably.

[0110] As used herein, an "effective amount," "effective dose," or "effective quantity" of a drug, compound, or pharmaceutical composition is an amount sufficient to affect any one or more beneficial or desired outcomes. In a more specific sense, an effective amount prevents, alleviates, or improves symptoms of a disease, and / or prolongs the survival of an individual undergoing treatment. For preventative use, beneficial or desired outcomes include, but are not limited to: eliminating or reducing the risk of disease, reducing the severity of disease, or delaying the onset of disease, including the disease, its complications, and biochemical, histological, and / or behavioral symptoms of intermediate pathological phenotypes that occur during disease development. For therapeutic use, beneficial or desired outcomes include, but are not limited to, clinical outcomes such as: reducing one or more symptoms of a disease (e.g., cancer, including, but not limited to, solid tumors), reducing the dosage of other agents required to treat the disease, enhancing the effect of another drug treatment, and / or delaying the progression of cancer in a patient. An effective amount may be administered in one or more doses. For the purposes of this disclosure, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve preventative or therapeutic treatment. As understood in clinical context, an effective dosage of a drug, compound, or drug composition may or may not be achieved when combined with another drug, compound, or drug composition. Therefore, "effective dosage" or "effective amount" can be considered in the context of administering one or more therapeutic agents, and if the desired result is achieved when combined with one or more other agents, then the single agent can be considered to have been administered in an effective amount.

[0111] "Individual" or "subject" refers to a mammal, with a non-limiting example being a human. Mammals also include, but are not limited to, farm animals (e.g., cows, pigs, horses, chickens, etc.), racing animals, pets, primates, horses, dogs, cats, mice, and rats.

[0112] As used herein, "vector" or "expression vector" means a construct capable of delivering and expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0113] As used herein, an "expression control sequence" is a nucleic acid sequence that guides the transcription of nucleic acids. The expression control sequence can be a promoter, such as a constitutive or inducible promoter; or an enhancer. The expression control sequence is operatively linked to the nucleic acid sequence to be transcribed.

[0114] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutical acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain its biological activity or stability and does not react with an individual's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers, such as phosphate-buffered saline solutions, water, emulsions (e.g., oil / water emulsions), and various types of wetting agents. For aerosol or parenteral administration, preferred diluents are phosphate-buffered saline (PBS) or physiological saline (0.9%). Compositions containing these carriers are formulated using known conventional methods.

[0115] As used herein, the term "kon" refers to the rate constant of antibody association with the antigen. Specifically, the rate constants (kon and koff) and equilibrium dissociation constants are measured using full-length antibodies and / or Fab antibody fragments (i.e., monovalent) and PD-1.

[0116] As used in this article, the term "koff" refers to the rate constant of antibody dissociation from the antibody / antigen complex.

[0117] The term "immunomodulator" refers to any substance capable of altering (e.g., suppressing, reducing, enhancing, strengthening, or stimulating) an immune response (as defined herein) or processing any component of the innate, humoral, or cellular immune system of a host mammal. Therefore, the term "immunomodulator" encompasses "immune effector cell enhancers" and "immunosuppressor cell inhibitors" as defined herein, as well as substances that affect other components of the mammalian immune system.

[0118] The term “immune response” refers to any detectable response of the host mammal’s immune system to a specific substance (such as an antigen or immunogen), such as innate immune responses (e.g., activation of the dopamine receptor signaling cascade), cell-mediated immune responses (e.g., responses mediated by T cells, such as antigen-specific T cells, and non-specific cells of the immune system), and humoral immune responses (e.g., responses mediated by B cells, such as the production and secretion of antibodies into plasma, lymph, and / or tissue fluid).

[0119] The term "immunogenicity" refers to the ability of a substance to elicit, trigger, stimulate, or induce an immune response or to improve, enhance, increase, or prolong a pre-existing immune response in response to a specific antigen, whether alone or in connection with a carrier, in the presence or absence of an adjuvant.

[0120] The term "immunosuppressive cell inhibitor" or "ISC inhibitor" refers to a substance that can reduce or suppress the number or function of immunosuppressive cells in mammals. Examples of immunosuppressive cells include regulatory T cells ("Tregs"), bone marrow-derived suppressor cells, and tumor-associated macrophages.

[0121] In the context of administering substances to mammals, including humans, the terms "intradermal application," "id," or "administered via an intradermal route" refer to the delivery of a substance into the dermis of the mammalian skin. Mammalian skin consists of the epidermis, dermis, and subcutaneous layer. The epidermis is the outermost layer of the skin. The dermis (the middle layer of the skin) contains nerve endings, sweat glands and sebaceous glands, hair follicles, and blood vessels. The subcutaneous layer consists of fat and connective tissue that houses larger blood vessels and nerves. In contrast to intradermal application, "subcutaneous application" refers to the application of a substance into the subcutaneous layer, and "local application" refers to the application of a substance onto the skin surface.

[0122] The term “preventing” or “prevention” means (a) preventing the onset of a disease, or (b) delaying the onset of a disease or the onset of its symptoms.

[0123] The term "tumor-associated antigen" or "TAA" refers to an antigen specifically expressed by tumor cells or expressed by tumor cells at a higher frequency or density compared to non-tumor cells of the same tissue type. Tumor-associated antigens may be antigens not normally expressed by the host; they may be mutations, truncations, misfolds, or other aberrant expressions of molecules normally expressed by the host; they may be the same molecules as normally expressed molecules but expressed at abnormally high levels; or they may be expressed under abnormal conditions or environments. Tumor-associated antigens can be, for example, proteins or protein fragments, complex carbohydrates, gangliosides, haptens, nucleic acids, or any combination of these or other biomolecules.

[0124] The term "vaccine" refers to an immunogenic composition intended for administration to mammals to elicit an immune response against a specific antigen found in the mammal. Vaccines typically contain agents (known as "antigens" or "immunogens") that are similar to or derived from the target of the immune response, such as pathogenic microorganisms or tumor cells. Vaccines intended for the treatment of tumors (e.g., cancer) typically contain antigens derived from TAAs found on the target tumor and are capable of inducing immunogenicity against the TAAs on the target tumor.

[0125] The term "vaccine-based immunotherapy regimen" refers to a treatment regimen in which a vaccine is administered in combination with one or more immunomodulatory agents. The vaccine and immunomodulatory agents may be administered together in a single formulation or separately.

[0126] As used herein, the term "antigen-binding portion" (or simply "antibody portion") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., PD-1). Antigen-binding function of antibodies has been demonstrated to be performed by fragments of full-length antibodies. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bonds in the hinge region; (iii) Fd fragments, consisting of VH and CH1 domains; (iv) Fv fragments, consisting of the VL and VH domains of a single arm of the antibody; (v) dAb fragments, consisting of a VH domain; and (vi) separated complementarity-determining regions (CDRs). Furthermore, although the two domains VL and VH of the Fv fragment are encoded by independent genes, they can be conjugated using recombinant methods by enabling them to be produced as synthetic linkers in the form of a single protein chain, where the VL region pairs with the VH region to form a monovalent molecule (called a single-chain Fv (scFv)). These single-chain antibodies are also intended to be included within the “antigen-binding portion” of the term antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and are screened for utility in the same manner as intact antibodies.

[0127] As used herein, the term "human antibody" is intended to include antibodies in which both the framework region and the CDR region of the variable region are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, then the constant region is also derived from a human germline immunoglobulin sequence. Human antibodies of this disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced through random or site-directed mutagenesis in vitro, or through somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human framework sequence.

[0128] The term "human monoclonal antibody" refers to an antibody that exhibits single-binding specificity, where both the framework region and the CDR region of the variable region are derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody is produced by a hybridoma comprising B cells obtained from a transgenic non-human animal (e.g., a transgenic mouse) fused with immortalized cells, the transgenic non-human animal having a genome containing both human heavy chain transgenes and light chain transgenes.

[0129] As used herein, the terms “humanized antibody” or “recombinant human antibody” include all human antibodies prepared, expressed, generated, or isolated in a recombinant manner, such as (a) antibodies isolated from transgenic or transchromosomally modified animals (e.g., mice) or hybridomas prepared therefrom (described further below); (b) antibodies isolated from host cells transgenic to express human antibodies, such as from transfected tumors; (c) antibodies isolated from recombinant, combined human antibody libraries; and (d) antibodies prepared, expressed, generated, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. These recombinant human antibodies have variable regions in which the frame region and CDR region are derived from human germline immunoglobulin sequences. However, in some embodiments, these recombinant human antibodies may be mutagenized in vitro (or in vivo somatic cell mutagenized when using transgenic animals with human Ig sequences), so the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that, although derived from and associated with human germline VH and VL sequences, may not be naturally present in the germline lineage of human antibodies in vivo.

[0130] As used in this article, “isotype” refers to the antibody class (e.g., IgM or IgG1) encoded by genes in the heavy chain constant region.

[0131] The phrases “antibody that recognizes an antigen” and “antibody that is specific to an antigen” are used interchangeably with the term “antibody that specifically binds to an antigen” in this document.

[0132] The term "human antibody derivative" refers to any modified form of a human antibody, such as a combination of an antibody with another drug or antibody.

[0133] The term "chimeric antibody" refers to an antibody whose variable region sequence originates from one species and whose constant region sequence originates from another species, such as an antibody whose variable region sequence originates from a mouse antibody and whose constant region sequence originates from a human antibody.

[0134] As used herein, the term "Kassoc" or "Ka" refers to the association rate of a specific antibody-antigen interaction, while the term "Kdis" or "Kd" refers to the dissociation rate of a specific antibody-antigen interaction. As used herein, the term "KD" refers to the dissociation constant, which is obtained as the ratio of Kd to KA (i.e., Kd / Ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods recognized in the art. Methods for determining antibody KD include the use of surface plasmon resonance, for example, using a biosensor system such as the Biacore (trademark) system.

[0135] As used herein, the term "high affinity" for IgG antibodies refers to an antibody's affinity for the target antigen of approximately 10. -8 M or lower, approximately 10-9 M or lower, or about 10 -10 M or lower KD. However, "high affinity" binding can vary for other antibody isotypes. For example, "high affinity" binding for IgM isotypes refers to binding with approximately 10 KD. -7 M or lower, approximately 10 -8 M or lower, or about 10 -9 M or lower KD.

[0136] As used herein, “about” means within an acceptable margin of error for a particular value as determined by one of ordinary skill in the art, the margin of error depending in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, “about” may mean within one or more standard deviations. Alternatively, “about” may mean a range of up to ±5%, ±10%, ±15%, or ±20%. Furthermore, particularly in biological systems or processes, the term may mean up to one order of magnitude or up to ±3 times the value. When a particular value is provided in this application and the claims thereof, unless otherwise stated, the meaning of “about” should be assumed to be within an acceptable margin of error for that particular value. References herein to “about” a value or parameter include (and description) embodiments of that value or parameter itself. For example, a description referring to “about X” includes a description of “X”. Numerical ranges include numerical values ​​that define the range. Alternatively or additionally, throughout this disclosure, values ​​expressed in range format should be interpreted flexibly, including not only the numerical values ​​whose boundaries are explicitly listed, but also all individual numerical values ​​or subranges encompassed within that range, as if each numerical value and subrange were explicitly listed. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted as including not only about 0.1% to about 5%, but also the individual values ​​(e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range, and such figures may be accurate to two decimal places. Unless otherwise indicated, the expression "about X to Y" has the same meaning as "about X to about Y". Similarly, unless otherwise indicated, the expression "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z".

[0137] As used herein, the term "conserved sequence modification" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing an amino acid sequence. These conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of this disclosure using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conserved amino acid substitution is the substitution of an amino acid residue by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include the following amino acids: those with basic side chains (e.g., lysine, arginine, histidine), those with acidic side chains (e.g., aspartic acid, glutamic acid), those with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), those with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), those with β-branched side chains (e.g., threonine, valine, isoleucine), and those with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR region of the antibodies disclosed herein may be substituted with other amino acid residues from the same side chain family, and the altered antibodies may be tested using the functional assays described herein to determine the retained function (i.e., the functions shown in (c) to (I) above).

[0138] The term “treatment” or “therapy” refers to the application of an active agent for the purpose of curing, healing, slowing, alleviating, altering, remedying, reducing, improving or affecting a symptom (e.g., disease), symptoms of a symptom, or preventing or delaying the onset of symptoms, complications, biochemical markers of a disease, or otherwise, in a statistically significant manner, inhibiting or suppressing the further development of a disease, symptom or condition.

[0139] As used herein, an “adverse event” (AE) is any unfavorable and generally unexpected, or even undesirable, sign (including abnormal laboratory test results), symptom, or illness associated with the use of a medical treatment. For example, an adverse event may be associated with activation of the immune system in response to treatment or expansion of immune system cells (e.g., T cells). Medical treatment may result in one or more associated AEs, and each AE may have the same or different severity. The term “methods that can “modify adverse events” refers to treatment regimens that reduce the incidence and / or severity of one or more AEs associated with the use of different treatment regimens.

[0140] As used herein, the term "drug" means any compound having the desired biological activity and reactive functional groups that can be used to prepare the conjugates of this disclosure. Biological activity includes activities for the diagnosis, cure, amelioration, treatment, or prevention of diseases in humans or other animals. Therefore, compounds covered by the term "drug" include those identified in official national pharmacopoeias and, for example, the U.S. Homeopathic Pharmacopeia or the National Formulary or any of their supplements, provided they have the desired reactive functional groups. Exemplary drugs are described in the Physician's Desk Reference (PDR) and the Orange Book maintained by the U.S. Food and Drug Administration (FDA).

[0141] As used herein, the term "biologic drug" refers to any compound or protein having the desired biological activity and reactive functional groups that can be used to prepare the conjugates of this disclosure. Biological activity includes activity for the diagnosis, cure, amelioration, treatment, or prevention of diseases in humans or other animals. Therefore, the compound or protein referred to by the term "biologic drug" includes drugs identified in official national pharmacopoeias and, for example, the Purple Book maintained by the U.S. Food and Drug Administration (FDA), provided it has the desired reactive functional groups.

[0142] As used in this article, the term HCC refers to hepatocellular carcinoma, which is a cancer that originates in the liver. It differs from "secondary" liver cancer, which is cancer that spreads to the liver from other organs.

[0143] The term "cytotoxic agent" refers to a substance that inhibits or blocks cell expression activity, cell function, and / or causes cell damage. The term includes radioactive isotopes, chemotherapeutic agents, and toxins, such as small molecule toxins or enzyme-active toxins (including fragments and / or variants) derived from bacteria, fungi, plants, or animals. Examples of cytotoxic agents include, but are not limited to: auristatin (e.g., auristatin E, auristatin F, MMAE, and MMAF), chlortetracycline, metotanol, ricin, ricin A chain, cobustatin, dokamicin, dorastatin, adriamycin, daunorubicin, paclitaxel, cisplatin, CC1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracnosediketone, actinomycin, and diphtheria toxin. The drugs include toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, α-Sarcina, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crocotin, calicheamicin, Sapaonaria officinalis inhibitor, glucocorticoids and other chemotherapeutic agents, and radioactive isotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or Bi213, P32 and Lu (including Lu177). Antibodies can also bind to prodrug-activating enzymes, which can convert prodrugs into their active form.

[0144] Figure 1(a) shows the complete heavy chain of P1801, including three CDRs, a variable domain, and a constant region. It also includes the heavy chain signal peptide, which will eventually be cleaved by the host cell. The complete heavy chain sequence is represented as SEQ ID NO: 1. Figure 1(b) shows the complete light chain of P1801, including three CDRs, a variable domain, and a constant region. It also includes the light chain signal peptide, which will eventually be cleaved by the host cell. The complete light chain sequence is represented as SEQ ID NO: 2. Figure 1(c) shows the heavy chain of P1801 without the heavy chain signal peptide (SEQ ID NO: 3). Figure 1(d) shows the light chain of P1801 without the light chain signal peptide (SEQ ID NO: 4). Figure 1(f) shows the complete heavy chain variable region of P1801 (SEQ ID NO: 5). Figure 1(g) shows the complete light chain variable region of P1801 (SEQ ID NO: 6). Figure 1(h) shows the heavy chain constant region of P1801 (SEQ ID NO: 7). Figure 1(i) shows the light chain constant region of P1801 (SEQ ID NO: 8). Figure 1(j) shows the heavy chain CDR1 of P1801 (SEQ ID NO: 9). Figure 1(k) shows the heavy chain CDR2 of P1801 (SEQ ID NO: 10). Figure 1(l) shows the heavy chain CDR3 of P1801 (SEQ ID NO: 11). Figure 1(m) shows the light chain CDR1 of P1801 (SEQ ID NO: 12). Figure 1(n) shows the light chain CDR2 of P1801 (SEQ ID NO: 13). Figure 1(o) shows the light chain CDR3 of P1801 (SEQ ID NO: 14). Figure 1(p) shows the heavy chain signal peptide of P1801 (SEQ ID NO: 15). Figure 1(q) shows the light chain signal peptide of P1801 (SEQ ID NO: 16). Figure 1(r) shows the hinge sequence located in the heavy chain (SEQ ID NO: 17). Figure 1(s) shows the alternative heavy chain CDR1 of P1801 (SEQ ID NO: 18). Figure 1(t) shows the alternative heavy chain CDR1 of P1801 (SEQ ID NO: 19). Figure 1(u) shows the alternative heavy chain CDR1 of P1801 (SEQ ID NO: 20). Figure 1(v) shows the alternative heavy chain CDR1 of P1801 (SEQ ID NO: 21). Figure 1(w) shows the alternative heavy chain CDR2 of P1801 (SEQ ID NO: 10). Figure 1(x) shows the alternative heavy chain CDR3 of P1801 (SEQ ID NO: 23).

[0145] As used herein, the term "P1801" refers to the anti-PD-1 monoclonal antibody of this disclosure shown in Figure 1. For example, the detailed amino sequences of the heavy chain / light chain and all CDRs or alternative CDRs are shown in... Figures 1(a) to 1(o) as well as Figures 1(a) to 1(o) As shown in Figure 1(s) (without signal peptide). Figure 1 represents a schematic of human P1801 with the alternative CDR shown in Figure 1(s).

[0146] As used herein, the terms “P1101,” “Ropeg,” and / or “polyethylene glycol-modified human interferon α2b” are used interchangeably. P1101 is known in the art, for example, see U.S. Patent Nos. 8,143,214 B2, 8,273,343, 8,617,532, and / or 8,106,160 B2, the contents of which are incorporated herein by reference in their entirety. See also U.S. Patent Application Nos. 20170326206A1, 20220152156A1, 20220362343A1, and / or 20230057788A1, the contents of which are incorporated herein by reference in their entirety. In short, for example, chemical formulas, methods of manufacture, and uses thereof are disclosed therein. For convenience, its structure is shown in… Figure 2 The diagram is simplified below. More specifically, interferon is the form that functions against P1101 in human individuals.

[0147] As used in this article, the term "mP1101" refers to a structure with the same polyethylene glycol and linker as P1101, but the difference is that the interferon is a mouse form that is functional in rodent individuals.

[0148] ii. Composition and molecular outline

[0149] peptide sequence

[0150] Figures 1(a) to 1(x) Examples of various amino acid sequences of P1801 are shown. Table 1 below shows the molecular characteristics of P1801.

[0151] Table 1

[0152]

[0153] In some embodiments, the antibodies of this disclosure comprise heavy chain variable regions from a specific germline heavy chain immunoglobulin gene and / or light chain variable regions from a specific germline light chain immunoglobulin gene.

[0154] In one embodiment, the nucleic acid encoding P1801 and the vector expressing these amino acids contain a nucleic acid molecule encoding a signal peptide (heavy chain SEQ ID NO: 15 and light chain SEQ ID NO: 16), wherein the signal peptide is located at the N-terminus of a variable domain of the heavy chain and / or light chain. Alternatively, in one embodiment, the nucleic acid encoding the light chain signal peptide does not include any introns. These signal peptides may be expressed first in host cells, subsequently lysed by the host cells, and then secreted or released into a solution outside the host cells, thereby generating P1801.

[0155] Nucleic acid molecules encoding antibody P1801

[0156] Another aspect of this disclosure relates to nucleic acid molecules encoding antibodies of this disclosure. Nucleic acids may be present in whole cells, cell lysates, or in partially purified or substantially pure forms. Nucleic acids are “isolated” or “substantially pure” when purified by standard techniques (including alkali / SDS treatment, CsCl clustering, column chromatography, agarose gel electrophoresis, and other techniques well known in the art) and separated from other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins). For example, DNA or RNA may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is an eDNA molecule.

[0157] The nucleic acids disclosed herein can be obtained using standard molecular biology techniques. For antibodies expressed from hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as further described below), the cDNA encoding the light and heavy chains of the antibody prepared from the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), the nucleic acids encoding the antibodies can be recovered from the library.

[0158] Once the DNA fragments encoding the VH and VL regions are obtained, these fragments can be further manipulated using standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding VL or VH can be operatively linked to another DNA fragment encoding a different protein, such as an antibody constant region or a flexible linker. The term "operatively linked" as used in this context means that the two DNA fragments are joined in such a way that the amino acid sequences encoded by both DNA fragments remain within the frame.

[0159] In one embodiment, isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably ligating the VH-encoding DNA to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art, and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant regions can be constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD, particularly the constant regions of IgG1 or IgG4. For Fab fragment heavy chain genes, the VH-encoding DNA is operably ligated to another DNA molecule encoding only the heavy chain CH1 constant region.

[0160] In one embodiment, isolated DNA encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene) by operably ligating the VL-encoding DNA to another DNA molecule encoding the light chain constant region CL. The sequences of the human light chain constant region genes are known in the art, and DNA fragments covering these regions can be obtained by standard PCR amplification. The light chain constant region can be a κ or λ constant region, or a κ constant region.

[0161] In order to generate the scFv gene, the DNA segments encoding VH and VL are operatively linked to another segment encoding a flexible adapter (e.g., encoding the amino acid sequence (Gly4-Ser)3), so that the VH and VL sequences can be expressed as adjacent single-stranded proteins, wherein the VL and VH regions are joined by a flexible adapter.

[0162] iii. Preparation method

[0163] This disclosure also provides methods for generating, selecting, and preparing anti-PD-1 antibodies. The antibodies of this disclosure can be prepared using procedures known in the art. In some embodiments, the antibodies can be prepared recombinantly and expressed using any method known in the art.

[0164] clone

[0165] Cloning vectors can be constructed using known techniques or selected from a wide range of cloning vectors available in the relevant field. While the chosen cloning vector may vary depending on the intended host cell, the final cloning vector is generally self-replicating, has a single target for a specific restriction endonuclease, and / or carries a gene that can be used to select clones containing the vector. Suitable examples include plasmids and bacterial viruses such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors (e.g., pSA3 and pAT28). These, and many other commercially available cloning vectors, are available from various vendors.

[0166] This disclosure further provides expression vectors. Expression vectors are generally replicable polynucleotide constructs containing polynucleotides according to this disclosure. This implies that the expression vector, whether as a free genome or as part of the chromosomal DNA as a whole, must be replicable in the host cell. Suitable expression vectors include, but are not limited to, plasmids, viral vectors (including adenoviruses, adeno-associated viruses, and retroviruses), granules, and the expression vectors disclosed in PCT Publication WO 87 / 04462. Vector components generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; and suitable transcriptional control elements (e.g., promoters, enhancers, and terminators). For expression (i.e., translation), one or more translational control elements are typically also required, such as ribosome binding sites, translation initiation sites, and stop codons.

[0167] Vectors containing the polynucleotide of interest can be introduced into host cells via any of a variety of suitable methods, including electroporation; transfection with calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microbolite bombardment; liposome transfection; and infection (e.g., where the vector is an infectious agent such as vaccinia virus). The choice of vector or polynucleotide depends on the characteristics of the host cell.

[0168] This disclosure also provides host cells comprising any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DNA can be used to isolate genes encoding the antibody, peptide, or protein of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. See also PCT Publication WO87 / 04462. Suitable non-mammalian host cells include prokaryotes (e.g., *Escherichia coli* or *Bacillus subtilis*) and yeasts (e.g., *Saccharomyces cerevisiae*, *Saccharomyces pombe*, or *Kluyveromyces lactis*). In one aspect, the level of cDNA expression in the host cell is approximately 5, 10, or 20 times higher than the expression level of the corresponding endogenous antibody or protein of interest (if present) in the host cell. In one embodiment, P1801 can be prepared using Chinese hamster ovary (CHO) DG44 cells, which are dhfr gene-deficient cell lines (dhfr-) derived from the CHO cell lineage.

[0169] Screening for host cells that specifically bind to PD-1 or its domain can be influenced by immunoassays or fluorescence-activated cell sorting (FACS). Cells overexpressing the desired antibody or protein can be identified.

[0170] Expression vectors can be used to guide the expression of anti-PD-1 antibodies. Those skilled in the art are familiar with the administration of expression vectors used to obtain the expression of exogenous proteins in vivo. See, for example, U.S. Patents 6,436,908, 6,413,942, and 6,376,471, all of which are incorporated herein by reference in their entirety. Administration of the expression vector includes local or systemic administration, including injection, oral administration, particle gun or catheter administration, and local administration. In another embodiment, the expression vector is administered directly to the sympathetic trunk or ganglion, or to the coronary artery, atrium, ventricle, or pericardium.

[0171] The vector can be transfected into host cells and stored in a Master Cell Bank. Typically, one or two Master Cell Bank (MCB) vials are removed from a liquid nitrogen container. After thawing at approximately 37°C, the MCB vials are transferred to 125 ml Tri-forest shaking flasks containing pre-warmed Dynamis alkaline medium in a biosafety cabinet. Cells can then be cultured in an INFORS incubator at approximately 37°C with shaking.

[0172] Subculturing of the inoculum: After approximately 72 to 96 hours of cell growth, transfer the initial inoculum to a 500 ml Tri-forest shaking flask containing Dynamis alkaline medium and approximately 30 ml of cells in a biosafety cabinet. This subculturing can be repeated once or multiple times as needed.

[0173] After cell growth, the cell culture medium can be seeded into SUB50 containing alkaline medium LC100B. Cell density is determined by sampling the culture and measuring using a Beckman Vi-Cell counter. The pH of the batch-fed cell culture is controlled. Dissolved oxygen (DO) is maintained at approximately 45% while agitating. Feed medium and components can be added individually based on a fixed schedule. Antifoaming emulsion is added as needed. The seeding cell density can be approximately 1.5 × 10⁻⁶ cells / year. 6 Cells / mL. Once peak cell density is reached, the cell culture is harvested. After harvesting, the batch-fed culture can be filtered one or more times. After filtration, the culture is collected in a 50 L storage bag and stored at approximately 4°C. Methods known in the art for purifying antibodies and other proteins include, for example, those described in U.S. Patent Publications 2008 / 0312425, 2008 / 0177048, and 2009 / 0187005, each of which is incorporated herein by reference in its entirety.

[0174] General characteristics

[0175] P1801 blocks the interaction between the immune checkpoint PD-1 and its ligand PD-L1 to disrupt immunosuppressive mechanisms. P1801 belongs to the IgG4 subclass of human antibodies and has been shown to exhibit unique properties. Human IgG4 antibodies can be functionally monovalent in vivo because they may exchange half-molecules (one heavy chain and one light chain) with each other, a process called Fab arm exchange. The Fab domain of P1801 is linked to the Fc via a hinge region with the amino acid sequence ESKYGPPCPPCP (SEQ ID NO: 17) located within the heavy chain constant region. This hinge region has an S288P mutation to reduce Fab arm exchange. IgG4 exhibits low affinity for complement component 1q (C1q) and the Fc receptor, causing weak induction of complement toxicity and antibody-dependent cell activation. These properties of IgG4 are preferred in cancer immunotherapy when host effector functions are not desired.

[0176] In another embodiment, P1801 may retain all variable regions and change the IgG type to IgG1, IgG2, IgG3 or other types.

[0177] Hybridoma screening

[0178] Anti-PD1 antibodies (such as P1801) can also be produced through hybridoma selection. For example, murine antibodies are selected as one of the best clones due to their high binding affinity in ELISA. They specifically bind to human PD1 and can block the binding of PD1 to PDL1 / PDL2. Furthermore, the murine formula of P1801 is humanized and formatted as the human immunoglobulin subtype IgG4 because IgG4 exhibits less immune response, such as ADCC and CDC, compared to other subtypes IgG1 and IgG3. The DNA sequence of P1801 is codon-optimized for expression. The expression vector for P1801 can be synthesized using DNA 2.0.

[0179] Phage display

[0180] In some embodiments, antibodies can be prepared and selected using phage display technology. See, for example, U.S. Patents 5,565,332, 5,580,717, 5,733,743, and 6,265,150, all of which are incorporated herein by reference in their entirety. Alternatively, phage display technology can be used to generate human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene lineages from unimmunized donors. According to this technology, the antibody V domain gene frame is cloned into the major or minor coat protein genes of filamentous phages, such as M13 or fd, and displayed as a functional antibody fragment on the surface of phage particles. Because filamentous particles contain a single-stranded DNA copy of the phage genome, selection based on antibody functional characteristics also leads to the selection of genes encoding antibodies exhibiting those characteristics. Thus, phages mimic some characteristics of B cells. Phage display can be performed in various forms. Several sources of the V gene segment can be used for phage display. Known techniques have reported the isolation of a range of different anti-oxazolinone antibodies from a small, randomized library of V genes derived from the spleen of immunized mice. Lineages of V genes from human donors can be constructed, and antibodies against a range of different antigens, including self-antigens, can be isolated largely using techniques known in the field. In natural immune responses, mutations in antibody genes rapidly aggregate (somatic hypermutation). Some of the introduced changes confer higher affinity and preferentially replicate and differentiate into B cells displaying high-affinity surface immunoglobulins during subsequent antigen stimulation. This natural process can be simulated using a technique known in the field called “chain shuffling.” In this method, the affinity of “primary” human antibodies obtained via phage display is improved by sequentially replacing the heavy and light chain V region genes with a lineage (lineage) of naturally occurring variants of the V domain genes obtained from a never-immunized donor. This technique allows for the production of antibodies and antibody fragments with affinities in the pM-nM range. Strategies for preparing large phage antibody lineages (also known as "the mother-of-all libraries") have been described and are known in the field. Genetic shuffling can also be used to derive human antibodies from rodent antibodies, wherein the human antibodies have similar affinity and specificity to the starting rodent antibody. According to this method (also known as "epitope imprinting"), the heavy or light chain V domain genes of rodent antibodies obtained by phage display technology are replaced with a lineage of human V domain genes, producing rodent-human chimeras. Selection of the antigen allows for the isolation of human variable regions capable of restoring functional antigen-binding sites, i.e., the selection of epitope-controlled (imprinted) conjugates. When this process is repeated to replace the remaining rodent V domains, human antibodies are obtained (see PCT Publication WO 93 / 06213).Unlike traditional methods of humanizing rodent antibodies through CDR grafting, this technology provides fully human antibodies that do not contain rodent-derived frameworks or CDR residues.

[0181] In some embodiments, antibodies may be prepared using hybridoma technology. It is considered that any mammalian individual, including humans, or antibody-producing cells derived from them, can be manipulated to serve as the basis for generating mammalian (including human) hybridoma cell lines. As further described herein, the route and schedule of host animal immunization generally conform to techniques known for antibody stimulation and production. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantarily, and / or intradermally with an immunogen, including those described herein.

[0182] Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using general somatic cell hybridization techniques. Available myeloma cell lines can be used for hybridization. Generally, this technique involves fusing myeloma cells with lymphocytes using a fusion accelerant such as polyethylene glycol or by an electrical method well known to those skilled in the art. After fusion, the cells are separated from the fusion medium and grown in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate unhybridized parental cells. Any culture medium supplemented with serum or without serum as described herein can be used to culture hybridomas that secrete monoclonal antibodies. As an alternative to cell fusion techniques, EBV immortalized B cells can be used to generate the PD-1 monoclonal antibody disclosed herein. Hybridomas or other immortalized B cells can be expanded and subcloned as needed, and the antiimmunogen activity of the supernatant can be determined by conventional immunoassay procedures, such as radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay.

[0183] Hybridomas that can be used as antibody sources encompass all derivatives and daughter cells of parental hybridomas that produce monoclonal antibodies that specifically or preferentially bind to PD-1 or a portion thereof.

[0184] Hybridomas producing these antibodies can be grown in vitro or in vivo using known procedures. Monoclonal antibodies can be isolated from culture media or body fluids, as needed, using conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration. Undesired activity (if present) can be removed, for example, by passing the formulation through an adsorbent made of an immunogen linked to a solid phase, and eluting or releasing the desired antibody from the immunogen. Immunizing a host animal with a PD-1 peptide or a fragment containing the target amino acid sequence can generate an antibody population (e.g., monoclonal antibodies). The PD-1 peptide or fragment containing the target amino acid sequence is used with a bifunctional agent or derivatizer, such as maleic benzoyl sulfosuccinimide (via cysteine ​​residue), N-hydroxysuccinimide (via lysine residue), glutaraldehyde, succinic anhydride, SOCl2, or R'N═C═NR (where R and R' are different alkyl groups), to bind to a protein that is immunogenic in the species to be immunized (e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor).

[0185] In one embodiment, the P1801 of interest (monoclonal or polyclonal) can be sequenced, and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in a vector within host cells, which can then be amplified and frozen for future use. The generation of recombinant monoclonal antibodies in cell cultures can be achieved by cloning antibody genes from B cells using methods known in the art. See, for example, U.S. Patent No. 7,314,622.

[0186] Humanization

[0187] In some embodiments, the polynucleotide sequence can be used for genetic manipulation to “humanize” the antibody or to improve its affinity or other characteristics. Antibodies can also be tailored for, for example, dogs, cats, primates, equines, and bovine animals.

[0188] In some embodiments, fully human antibodies can be obtained using commercially available mice engineered to express specific human immunoglobulins. Humanized or human antibodies can also be produced using transgenic animals designed to produce a more desirable (e.g., fully human) or more robust immune response. Examples of such technologies include Xenomouse (trademark) from Abgenix, Inc., and HuMAb-Mouse (trademark) and TC Mouse (trademark) from Medarex, Inc.

[0189] Antibodies can also be prepared recombinantly as follows: First, antibodies and antibody-producing cells are isolated from a host animal, the gene sequence is obtained, and the antibody is recombinantly expressed in host cells (e.g., CHO cells) using the gene sequence. Another method is to express the antibody sequence in plants (e.g., tobacco) or transgenic latex. Methods for recombinantly expressing antibodies in plants or latex have been disclosed. Methods for preparing antibody derivatives such as domains and single chains are also known in the art.

[0190] Antibodies specific to PD-1 can also be isolated using immunoassays and flow cytometry sorting techniques such as fluorescence activated cell sorting (FACS).

[0191] DNA encoding monoclonal antibodies can be easily isolated and sequenced using routine procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of monoclonal antibodies). Hybridoma cells serve as a source of this type of DNA. Once isolated, the DNA can be placed in an expression vector (e.g., the expression vector disclosed in PCT Publication WO 87 / 04462) and subsequently transfected into host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not additionally produce immunoglobulins, to synthesize monoclonal antibodies in recombinant host cells. See, for example, PCT Publication WO 87 / 04462. The DNA can also be modified, for example, by replacing homologous murine sequences with coding sequences for constant domains of the human heavy and light chains, or by covalently attaching all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. In this way, “chimeric” or “hybrid” antibodies with the binding specificity of the PD-1 monoclonal antibody described herein are prepared.

[0192] Antibody fragments can be generated by the recombinant methods described above or by chemical synthesis, utilizing the protein degradation or other degradation of antibodies. Antibody peptides (especially shorter peptides of up to about 50 amino acids) can be prepared by chemical synthesis. Methods of chemical synthesis are known in the art and are commercially available. For example, antibodies can be produced using an automated peptide synthesizer employing a solid-phase method. See also U.S. Patents 5,807,715, 4,816,567, and 6,331,415, all of which are incorporated herein by reference in their entirety.

[0193] In some embodiments, the antibody that preferentially binds to PD-1 or its antigen-binding portion may have heavy chain complementarity-determining regions 1 (VHCDR1), 2 (VHCDR2), and / or 3 (VHCDR3) obtained (e.g., derived, identified, or copied) from the heavy chain variable region of the P1801 antibody (e.g., the sequence listed in SEQ ID NO: 5); and light chain complementarity-determining regions 1 (VLCDR1), 2 (VLCDR2), and / or 3 (VLCDR3) obtained from the light chain variable region of the P1801 antibody (e.g., the sequence listed in SEQ ID NO: 6). Those skilled in the art, taking into account the heavy chain variable region sequences or the light chain variable region sequences, will be able to determine all six CDR sequences based on the methods described herein or known in the art.

[0194] In one embodiment, analysis of the P1801 antibody or its antigen-binding portion that preferentially binds to PD-1, based on the Kabat assay, shows that P1801 comprises: a heavy chain complementarity-determining region 1 (VHCDR1) comprising the amino acid sequence listed in SEQ ID NO: 9, a heavy chain complementarity-determining region 2 (VHCDR2) comprising the amino acid sequence listed in SEQ ID NO: 10, and / or a heavy chain complementarity-determining region 3 (VHCDR3) comprising the amino acid sequence listed in SEQ ID NO: 11; and a light chain complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence listed in SEQ ID NO: 12, a light chain complementarity-determining region 2 (VLCDR2) comprising the amino acid sequence listed in SEQ ID NO: 13, and / or a light chain complementarity-determining region 3 (VLCDR3) comprising the amino acid sequence listed in SEQ ID NO: 14.

[0195] In another embodiment, based on the Kabat assay, analysis of the P1801 antibody or its antigen-binding portion that preferentially binds to PD-1 showed that P1801 comprises: a heavy chain complementarity-determining region 1 (VHCDR1) comprising the amino acid sequence listed in SEQ ID NO: 18, a heavy chain complementarity-determining region 2 (VHCDR2) comprising the amino acid sequence listed in SEQ ID NO: 10, and / or a heavy chain complementarity-determining region 3 (VHCDR3) comprising the amino acid sequence listed in SEQ ID NO: 11; and a light chain complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence listed in SEQ ID NO: 12, a light chain complementarity-determining region 2 (VLCDR2) comprising the amino acid sequence listed in SEQ ID NO: 13, and / or a light chain complementarity-determining region 3 (VLCDR3) comprising the amino acid sequence listed in SEQ ID NO: 14.

[0196] In another embodiment, based on the Chothia assay, analysis of the P1801 antibody or its antigen-binding portion that preferentially binds to PD-1 showed that P1801 comprises: a heavy chain complementarity-determining region 1 (VHCDR1) comprising the amino acid sequence listed in SEQ ID NO: 9, a heavy chain complementarity-determining region 2 (VHCDR2) comprising the amino acid sequence listed in SEQ ID NO: 10, and / or a heavy chain complementarity-determining region 3 (VHCDR3) comprising the amino acid sequence listed in SEQ ID NO: 20; and a light chain complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence listed in SEQ ID NO: 12, a light chain complementarity-determining region 2 (VLCDR2) comprising the amino acid sequence listed in SEQ ID NO: 13, and / or a light chain complementarity-determining region 3 (VLCDR3) comprising the amino acid sequence listed in SEQ ID NO: 14.

[0197] In another embodiment, based on the Chothia method, analysis of the P1801 antibody or its antigen-binding portion that preferentially binds to PD-1 showed that P1801 includes: a heavy chain complementarity-determining region 1 (VHCDR1) comprising the amino acid sequence listed in SEQ ID NO: 18, a heavy chain complementarity-determining region 2 (VHCDR2) comprising the amino acid sequence listed in SEQ ID NO: 10, and / or a heavy chain complementarity-determining region 3 (VHCDR3) comprising the amino acid sequence listed in SEQ ID NO: 20; and a light chain complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence listed in SEQ ID NO: 12, a light chain complementarity-determining region 2 (VLCDR2) comprising the amino acid sequence listed in SEQ ID NO: 13, and / or a light chain complementarity-determining region 3 (VLCDR3) comprising the amino acid sequence listed in SEQ ID NO: 14.

[0198] In one embodiment, analysis of the P1801 antibody or its antigen-binding portion that preferentially binds to PD-1, based on the Chothia assay, shows that P1801 comprises: a heavy chain complementarity-determining region 1 (VHCDR1) comprising the amino acid sequence listed in SEQ ID NO: 19, a heavy chain complementarity-determining region 2 (VHCDR2) comprising the amino acid sequence listed in SEQ ID NO: 10, and / or a heavy chain complementarity-determining region 3 (VHCDR3) comprising the amino acid sequence listed in SEQ ID NO: 20; and a light chain complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence listed in SEQ ID NO: 12, a light chain complementarity-determining region 2 (VLCDR2) comprising the amino acid sequence listed in SEQ ID NO: 13, and / or a light chain complementarity-determining region 3 (VLCDR3) comprising the amino acid sequence listed in SEQ ID NO: 14.

[0199] In another embodiment, based on the Chothia assay, analysis of the P1801 antibody or its antigen-binding portion that preferentially binds to PD-1 showed that P1801 comprises: a heavy chain complementarity-determining region 1 (VHCDR1) comprising the amino acid sequence listed in SEQ ID NO: 21, a heavy chain complementarity-determining region 2 (VHCDR2) comprising the amino acid sequence listed in SEQ ID NO: 10, and / or a heavy chain complementarity-determining region 3 (VHCDR3) comprising the amino acid sequence listed in SEQ ID NO: 20; and a light chain complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence listed in SEQ ID NO: 12, a light chain complementarity-determining region 2 (VLCDR2) comprising the amino acid sequence listed in SEQ ID NO: 13, and / or a light chain complementarity-determining region 3 (VLCDR3) comprising the amino acid sequence listed in SEQ ID NO: 14.

[0200] In one embodiment, analysis of the P1801 antibody or its antigen-binding portion that preferentially binds to PD-1, based on the Chothia assay, shows that P1801 comprises: a heavy chain complementarity-determining region 1 (VHCDR1) comprising the amino acid sequence listed in SEQ ID NO: 19, a heavy chain complementarity-determining region 2 (VHCDR2) comprising the amino acid sequence listed in SEQ ID NO: 10, and / or a heavy chain complementarity-determining region 3 (VHCDR3) comprising the amino acid sequence listed in SEQ ID NO: 11; and a light chain complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence listed in SEQ ID NO: 12, a light chain complementarity-determining region 2 (VLCDR2) comprising the amino acid sequence listed in SEQ ID NO: 13, and / or a light chain complementarity-determining region 3 (VLCDR3) comprising the amino acid sequence listed in SEQ ID NO: 14.

[0201] In another embodiment, based on the Chothia assay, analysis of the P1801 antibody or its antigen-binding portion that preferentially binds to PD-1 showed that P1801 comprises: a heavy chain complementarity-determining region 1 (VHCDR1) comprising the amino acid sequence listed in SEQ ID NO: 21, a heavy chain complementarity-determining region 2 (VHCDR2) comprising the amino acid sequence listed in SEQ ID NO: 10, and / or a heavy chain complementarity-determining region 3 (VHCDR3) comprising the amino acid sequence listed in SEQ ID NO: 11; and a light chain complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence listed in SEQ ID NO: 12, a light chain complementarity-determining region 2 (VLCDR2) comprising the amino acid sequence listed in SEQ ID NO: 13, and / or a light chain complementarity-determining region 3 (VLCDR3) comprising the amino acid sequence listed in SEQ ID NO: 14.

[0202] iv. Characteristics

[0203] At least two known FDA-approved human monoclonal antibodies against PD1 have been approved: pembrolizumab (trademark Keytruda) and nivolumab (trademark OPDIVO). Both can treat a variety of diseases, such as certain types of cancer. Furthermore, the epitopes on hPD1 for both pembrolizumab and nivolumab are known in their respective fields.

[0204] In one embodiment, as shown in FIG8, for example, P1801 of this disclosure binds to the same PD1 epitope as commercially available anti-PD1 monoclonal antibodies (e.g., pembrolizumab and / or nivolumab), completely overlapping. In another embodiment, P1801 binds to substantially the same PD1 epitope as commercially available anti-PD1 monoclonal antibodies (e.g., pembrolizumab and / or nivolumab), or has substantially overlapping regions. In yet another embodiment, P1801 partially binds to the same PD1 epitope region as commercially available anti-PD1 monoclonal antibodies (e.g., pembrolizumab and / or nivolumab). Surprisingly and unexpectedly, although P1801 partially overlaps with the PD1 epitope compared to pembrolizumab and / or nivolumab, indicating a weaker characterization, the kinetic properties of P1801 are substantially the same as, or even better than, pembrolizumab and / or nivolumab.

[0205] In yet another embodiment, P1801 only partially covers PD1, but surprisingly, its performance is at least comparable to that of existing commercially available anti-PD1 monoclonal antibodies.

[0206] In one embodiment, this disclosure discloses an anti-human PD1 monoclonal antibody (P1801) whose properties (e.g., molecular characteristics and / or cancer therapeutic efficacy) are substantially similar to those of commercially available anti-PD1 monoclonal antibodies (e.g., pembrolizumab and / or nivolumab). Exemplary features and properties include, but are not limited to, binding strength against PD1 (e.g., as measured by KD), stability / shelf life, and / or ability to treat cancer. Figure 3 This represents an instance in a mouse model.

[0207] In another embodiment, this disclosure discloses an anti-human PD1 monoclonal antibody (P1801) that exhibits significantly superior properties (e.g., molecular characteristics and / or cancer therapeutic efficacy) compared to commercially available anti-PD1 monoclonal antibodies. Exemplary features and properties include, but are not limited to, binding strength against PD1 (e.g., as measured by KD), stability / shelf life, and / or the ability to treat multiple types of cancer.

[0208] In another embodiment, this disclosure discloses a combination of P1101 and an anti-human PD1 monoclonal antibody (P1801) that exhibits significantly superior therapeutic and / or synergistic effects compared to commercially available anti-PD1 monoclonal antibodies alone in various aspects, such as molecular properties and / or cancer therapeutic efficacy. Exemplary features and properties include, but are not limited to, binding strength against PD1 (as measured by KD), stability / shelf life, and the ability to treat cancer and / or shrink tumor size.

[0209] Despite the alteration of the variable regions, those skilled in the art can modify the anti-PD1 antibodies or antigen-binding fragments thereof disclosed herein, such antibodies comprising at least a portion of one or more constant regions that have been deleted or otherwise altered to provide desired biochemical characteristics (e.g., increased tumor localization or decreased serum half-life compared to antibodies containing native or unaltered constant regions with substantially the same immunogenicity). In some aspects, the constant regions of the modified antibody will comprise human constant regions. Modification of constant regions compatible with the anti-PD1 molecules disclosed herein comprises the addition, deletion, or substitution of one or more amino acids in one or more domains. That is, the modified antibodies disclosed herein may comprise alterations or modifications to one or more of the three heavy chain constant domains (CH1, CH2, or CH3) and / or the light chain constant domain (CL). In some aspects, modified constant regions with partial or complete deletion of one or more of the domains are considered. In some respects, the modified antibody will include a domain-deleted construct or variant in which the entire CH2 domain has been removed. (CH2 construct). In some respects, the omitted constant region can be replaced by a short amino acid spacer (e.g., residues 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), which provides some of the molecular flexibility that would normally be conferred by the absence of a constant region.

[0210] Anti-PD1 antibodies or their antigen-binding fragments may be further modified to contain additional chemical moieties that are not normally proteins. These derived moieties can improve protein solubility, biological half-life, or absorption. The moieties can also reduce or eliminate any anticipated side effects of proteins, etc. The moieties may include one or more detectable markers or tags (e.g., biotin, reporter enzymes, colorimetric or fluorescent tags, solid carriers, or radiolabels) to facilitate the detection of antibodies or PD-1 in a sample.

[0211] In one embodiment, the complementary site of the anti-PD1 antibody or its antigen-binding fragment of this disclosure may contain amino acid residues from all VH and VL CDRs or only some CDRs (e.g., some CDRs may not participate in antigen binding). The complementary site against a specific antigen can be defined, for example, by scanning mutagenesis of amino acid residues within the antibody, particularly CDRs (e.g., alanine scanning mutagenesis), said amino acid residues considered to be exposed on the surface (e.g., determined by crystallographic modeling), and potentially involved in antigen binding. Subsequent evaluation of the mutant's binding to the antigen can determine whether the mutated amino acid position participates in antigen binding and thus forms part of the antibody's complementary site.

[0212] In another embodiment, complementary sites can be determined by high-resolution cryo-electron microscopy, which can produce intermolecular density maps to determine the binding distance between one or more CDRs and certain amino acids of the antigen.

[0213] In one embodiment, the anti-PD1 antibody or its antigen-binding fragment of the present disclosure comprises a heavy chain complementary site comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 9, 18, 19, or 21 (CDR1), 10 (CDR2), and 11 (CDR3). In another preferred embodiment, the anti-PD1 antibody or its antigen-binding fragment of the present disclosure comprises a light chain complementary site comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 12 (CDR1), 13 (CDR2), and 14 (CDR3). In one embodiment, the anti-PD1 antibody or its antigen-binding fragment of the present disclosure comprises: a heavy chain complementary site comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 9, 10, and 11, and a light chain complementary site comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 12, 13, and 14. In another embodiment, the anti-PD1 antibody or its antigen-binding fragment of the present disclosure comprises: a heavy chain complementary site comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 18, 10, and 11, and a light chain complementary site comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 12, 13, and 14. In another embodiment, the anti-PD1 antibody or its antigen-binding fragment of the present disclosure comprises: a heavy chain complementary site comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 19, 10, and 11, and a light chain complementary site comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 12, 13, and 14. In yet another embodiment, the anti-PD1 antibody or its antigen-binding fragment of the present disclosure comprises: a heavy chain complementary site comprising the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 21, 10 and 11, and a light chain complementary site comprising the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 12, 13 and 14.

[0214] Substitution of non-complementary amino acid residues

[0215] Non-complementary amino acids can be substituted through recombination to obtain variable light or heavy chain domains with properties similar to or altered from those of the germline variable domain. "Modified" variable domains also include amino acid deletions and substitutions. For example, N-terminal or C-terminal amino acid residues can be deleted in modified variable domains.

[0216] Non-complementary amino acid substitutions can be made, for example, to improve stability and / or reduce aggregation tendency. Poor stability can affect the ability of antigen-binding fragments to fold correctly, for example, when expressed in a recombinant manner, resulting in a portion of the expressed fragment being non-functional. Antibodies or their antigen-binding fragments with low stability may also be prone to forming potentially immunogenic aggregates, or have impaired affinity or shelf life. scFv peptides are particularly likely to exhibit problems with stability, solubility, expression, aggregation, degradation products, and overall manufacturability in bacterial and mammalian expression systems. For example, WO 2007 / 109254 discloses framework amino acid substitutions intended to improve the stability of, for example, the VH and / or VL domains in scFv peptides and / or reduce their aggregation tendency. Similarly, substitution of corresponding residues in the VH and VL domains in this case is expected to improve stability and / or reduce aggregation tendency.

[0217] v. A pharmaceutically acceptable carrier

[0218] The compositions used in this disclosure, whether P1801 alone, P1101 alone, or any combination thereof, may further comprise pharmaceutically acceptable carriers, excipients, or stabilizers, in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at certain doses and concentrations and may comprise: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethylbenzyl ammonium chloride; hexahydroxyquaternary ammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10). (Residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextran; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN (trademark), PLURONICS (trademark), or polyethylene glycol (PEG).

[0219] vi. dosage

[0220] In various embodiments, the concentration of anti-PD1 antibody administered is about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 16 mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg, or about 20 mg / kg.

[0221] vii. Combination

[0222] Anti-PD-1 antibodies can also be used in combination with other agents to enhance and / or supplement the effectiveness of antibody therapy for cancer, or administered separately, simultaneously, and / or sequentially. One such non-limiting example of an anti-PD1 monoclonal antibody is P1801, where the combination is P1801 + P1101. The anti-PD1 monoclonal antibody (e.g., P1801) or P1101 may be referred to as the first or second molecule in the combination. Alternatively, P1801 may be further optionally combined with another anti-PD1 monoclonal antibody (e.g., pembrolizumab, nivolumab, and / or cimipril).

[0223] In one embodiment, a murine anti-PD-1 antibody (e.g., RMP 1-14) can be used to replace P1801 in a mouse model. Alternatively, a mouse form of mP1101 with interferon can be used in a mouse model.

[0224] viii. Continuous and combined application

[0225] P1101 can be applied using various regimens and dosages previously reported in the field.

[0226] Continuous application

[0227] In addition, the length of time between consecutive administrations of one or more doses of P1101 or mP1101 to an individual can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks and / or 10 weeks. In another embodiment, P1801 may be continuously administered after the aforementioned continuous administration of P1101 or mP1101, wherein the length between one or more doses of P1801 administered to an individual is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks and / or 10 weeks.

[0228] Successive application in any of the above sequences (P1101 first or P1801 first) can provide a “synergistic effect” and demonstrate “synergistic effect”, that is, the effect achieved when the active ingredients are used together is greater than the sum of the effects produced by using the compounds separately.

[0229] Combined application

[0230] Combination therapy can also provide a “synergistic effect” and demonstrate “synergistic effect”, meaning that the effect achieved when the active ingredients are used together is greater than the sum of the effects produced by using the compounds separately. This disclosure also includes a synergistic effect that can be obtained when the active ingredients are: (1) co-formulated and simultaneously administered or delivered in unit dose formulations of the combination; (2) delivered alternately or in parallel as separate formulations; and / or (3) delivered via some other protocol. When delivered in alternating therapy, the synergistic effect can be obtained when the compounds are administered or delivered sequentially, for example, by different injections using separate syringes. Generally, during alternating therapy, each active ingredient is administered sequentially (i.e., continuously), while in combination therapy, two or more active ingredients are administered together in effective doses.

[0231] In one embodiment, the first and second molecules can be administered simultaneously to an individual by mixing them and applying them concurrently. Alternatively, the two macromolecules can be administered to the individual immediately in any given order, or sequentially and closely together in any given order. In another embodiment, one macromolecule can be administered first according to a given regimen, followed by the second macromolecule, which can then be administered according to a given regimen. In yet another embodiment, the regimen frequency and duration can vary in any length, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, and / or 31 days or longer. The first molecule can be P1101, and the second molecule can be P1801, or vice versa.

[0232] In some embodiments, the first molecule may be administered according to a first regimen during a first treatment period, and the second molecule may be administered according to a second independent regimen during a second independent treatment period, wherein the two regimens and / or treatment periods may be simultaneous, staggered, sequential, alternating, or timed in any way. For example, in the case where both molecules are administered once every 2 weeks, the two regimens may begin on the same day or different days (e.g., the second regimen may begin before or after the first regimen). In yet another example, where the first molecule is administered once every 5 days for 3 months and the second molecule is administered once every two weeks for 1 year, the two regimens may begin on the same day or different days (e.g., the second regimen may begin before or after the first regimen, on a day during the treatment period of the first molecule or a day not during the treatment period of the first molecule, or after the end of the treatment period of the first molecule). The dose and / or the time between each administration in the regimen may remain constant or vary. The first molecule may be P1101, and the second molecule may be an anti-PD1 monoclonal antibody, such as P1801. Alternatively, the first molecule can be an anti-PD1 monoclonal antibody, such as P1801, and the second molecule can be P1101.

[0233] In another embodiment, the first and second molecules can be administered alternately in any given order. In one embodiment, the first and second molecules can be administered in any fixed time interval order. Figure 4(a) illustrates such an example, where solid arrows indicate the administration of the first molecule to an individual, and dashed arrows indicate the administration of the second molecule, with each administration 3 days apart. Alternatively, the reverse can also be true, where solid arrows indicate the administration of the second molecule to an individual, and dashed arrows indicate the administration of the first molecule, with each administration 3 days apart. In another embodiment, an illustrative example is the administration of P1101 to an individual with solid arrows, and dashed arrows indicate the administration of an anti-PD1 monoclonal antibody, such as P1801. Alternatively, the reverse can also be true, where solid arrows indicate the administration of an anti-PD1 monoclonal antibody, such as P1801, to an individual, and dashed arrows indicate the administration of P1101.

[0234] In a non-limiting embodiment, the two molecules may be administered sequentially, but one molecule may initially be administered in a smaller number of doses (e.g., 3 doses) with a set time interval (e.g., 3 days) between each dose, followed by the second molecule administered in two, three, or four times the number of doses, also with a set time interval (e.g., 3 days) between each dose. A non-limiting example can be seen at the top of Figure 4(b). Another illustrative example is that a solid arrow indicates the administration of P1101 to an individual, and a dashed arrow indicates the administration of an anti-PD1 monoclonal antibody, such as P1801. The first molecule may be P1101, and the second molecule may be P1801, or vice versa.

[0235] In a non-limiting embodiment, the two molecules may be administered sequentially, but one molecule may initially be administered in the same number of doses (e.g., 6 doses) with a predetermined time interval (e.g., 3 days) between each dose, and then the second molecule may be administered in the same number of doses (e.g., 6 doses) with a predetermined time interval (e.g., 3 days) between each dose. The bottom of Figure 4(b) illustrates such a non-limiting example. Simply put, solid arrows indicate administration of P1101 to an individual, and dashed arrows indicate administration of an anti-PD1 monoclonal antibody, such as P1801. Alternatively, solid arrows indicate administration of an anti-PD1 monoclonal antibody, such as P1801, to an individual, and dashed arrows indicate administration of P1101.

[0236] In another embodiment, the first and second molecules may be administered alternately once each, but with an interval of 1 or 2 days between administrations. This regimen may be repeated two, three, four, or five times at fixed time intervals (e.g., every 3, 4, 5, or 6 days). Alternatively, either molecule may then be administered once at a single dose at a fixed time interval (e.g., 3, 4, 5, or 6 days). A non-limiting example of this is illustrated at the top of Figure 4(c). In one embodiment, solid arrows indicate administration of P1101 to an individual, and dashed arrows indicate administration of an anti-PD1 monoclonal antibody, such as P1801. Alternatively, solid arrows indicate administration of an anti-PD1 monoclonal antibody, such as P1801, to an individual, and dashed arrows indicate administration of P1101.

[0237] In one embodiment, the first and second molecules may be administered alternately once each, with an interval of 1 or 2 days between administrations. This regimen may be repeated two, three, four, or five times at fixed time intervals (e.g., every 3, 4, 5, or 6 days). A non-limiting example of this type is illustrated at the bottom of Figure 4(c). In one embodiment, a solid arrow indicates administration of P1101 to an individual, and a dashed arrow indicates administration of an anti-PD1 monoclonal antibody, such as P1801. Alternatively, a solid arrow indicates administration of an anti-PD1 monoclonal antibody, such as P1801, to an individual, and a dashed arrow indicates administration of P1101.

[0238] In a non-limiting embodiment, both molecules are administered simultaneously or on the same day, but one molecule may initially be administered at a smaller dose, followed by double, triple, or quadruple doses in subsequent second, third, fourth, or fifth administrations, while the second molecule is administered at a constant dose. Alternatively, the reverse may also be true, where the first molecule is administered at a constant dose, while the second molecule may initially be administered at a smaller dose, followed by double, triple, or quadruple doses in subsequent second, third, fourth, or fifth administrations. In one embodiment, the first molecule may be P1101, and the second molecule may be an anti-PD1 monoclonal antibody, such as P1801. Alternatively, the first molecule may be any anti-PD1 monoclonal antibody, such as P1801, and the second molecule may be P1101.

[0239] In another embodiment, the first molecule can be administered daily at a fixed dose for the first 13 days, and the second molecule can be administered starting on day 6, followed by 5 more administrations, all at 3-day intervals. Additionally, the first 4 administrations of the second molecule can have the same dosage, and the 5th administration can be twice the dosage of the previous 4. In the future, the 6th administration can be four times the dosage of the previous 4 administrations. Non-limiting examples may be... Figure 5As shown in the figure. In one embodiment, the first molecule administered may be P1101, and the second molecule may be an anti-PD1 monoclonal antibody, such as P1801. Alternatively, the first molecule may be an anti-PD1 monoclonal antibody, such as P1801, and the second molecule may be P1101.

[0240] In another embodiment, the first molecule may be administered ip or sc at intervals of 6, 7, or 8 days (QW×4 from day 1 to day 22), while the second molecule may be administered ip or sc on days 11, 13, 18, 20, 25, and 27 (BIW×2, from day 11 to day 27). Examples are shown in Figure 6 In one embodiment, the first molecule administered may be P1101, and the second molecule may be an anti-PD1 monoclonal antibody, such as P1801. Alternatively, the first molecule may be an anti-PD1 monoclonal antibody, such as P1801, and the second molecule may be P1101.

[0241] In one embodiment, the length between administering a single dose of P1801 or a single dose of P1101 to an individual can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks and / or 10 weeks or longer.

[0242] In another embodiment, the length between administering a single regimen P1801 and a single regimen P1101 to an individual can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks and / or 10 weeks or longer.

[0243] ix. Uses

[0244] In one embodiment, the use of an anti-PD1 monoclonal antibody in this disclosure enhances antigen-specific CD4. + or CD8 + T-cell activation enhances an individual's immunity to treat, suppress, and / or prevent cancers, such as solid tumors. Non-limiting examples of solid tumors include melanoma, hepatocellular carcinoma (HCC), and / or kidney tumors (renal cell carcinoma or RCC).

[0245] In another embodiment, the combination of P1801 and P1101, compared to P1801 alone, can further enhance an individual's immunity to treat, suppress, and / or prevent cancers, such as solid tumors. Non-limiting examples include melanoma, hepatocellular carcinoma (HCC), colorectal cancer (CRC), melanoma, and / or kidney tumors (renal cell carcinoma (RCC)).

[0246] In one embodiment, the P1801 and / or the combination of P1801 and P1101 disclosed in this invention can be used to treat, suppress, and / or prevent the following diseases:

[0247] Advanced melanoma or adjuvant therapy for melanoma

[0248] P1801 and / or a combination of P1801 with another molecule (e.g., P1101) may be used to treat individuals with unresectable or metastatic melanoma. This treatment may be further optionally combined with pembrolizumab and / or nivolumab.

[0249] Advanced hepatocellular carcinoma (HCC)

[0250] P1801 and / or a combination of P1801 with another molecule (e.g., P1101) may be used to treat individuals with hepatocellular carcinoma (HCC). The individual may have previously been treated with sorafenib. This treatment may optionally be further combined with cimiprimab, pembrolizumab, and / or nivolumab.

[0251] Treatment of renal cell carcinoma (RCC) or advanced RCC

[0252] P1801 and / or a combination of P1801 with another molecule (e.g., P1101) may be used as adjuvant therapy for individuals with renal cell carcinoma (RCC) who are at intermediate to high or high risk of recurrence after nephrectomy or after nephrectomy and resection of metastatic lesions. This treatment may optionally be further combined with cimiprimab, pembrolizumab, and / or nivolumab.

[0253] P1801 and / or a combination of P1801 with another molecule (e.g., P1101) may be used as first-line treatment for adult individuals with advanced renal cell carcinoma (RCC). This treatment may be further optionally combined with axitinib and / or cimiprimab, pembrolizumab, and / or nivolumab.

[0254] For adults with advanced melanoma

[0255] P1801, or in combination with another molecule (such as P1101), can be used to treat a type of skin cancer called melanoma that has spread or is not surgically removable (advanced melanoma). This therapy may be further optionally combined with cimiprimab, nivolumab, pembrolizumab, and / or ipilimumab.

[0256] For adults with melanoma, where the melanoma and affected lymph nodes have been surgically removed to prevent recurrence. individual

[0257] P1801, or in combination with another molecule (e.g., P1101), may be used to treat adult individuals with melanoma whose melanoma and affected lymph nodes have been surgically removed to prevent recurrence. This therapy may be further optionally combined with cimiprimab, pembrolizumab, and / or nivolumab.

[0258] For some individuals with advanced renal cell carcinoma (renal cell carcinoma)

[0259] When renal cell carcinoma has spread (advanced renal cell carcinoma) and some individuals with this cancer have not yet received treatment for advanced RCC, P1801, or in combination with another molecule (such as P1101), may be used to treat said individuals. This therapy may further be optionally combined with cimiprimab, nivolumab, pembrolizumab, and / or ipilimumab.

[0260] For individuals newly diagnosed with kidney cancer (renal cell carcinoma) that has already spread.

[0261] When renal cell carcinoma has spread (advanced renal cell carcinoma) and the patient has not yet received treatment for advanced RCC, P1801, or in combination with another molecule (such as P1101), can be used to treat the individual. This therapy may further be optionally combined with cimiprimab, nivolumab, pembrolizumab, and / or cabozantinib.

[0262] For individuals who have previously undergone treatment for advanced renal cell carcinoma (renal cell carcinoma)

[0263] When kidney cancer (renal cell carcinoma) has spread or grown after treatment with other cancer agents, P1801, or in combination with another molecule (such as P1101), can be used to treat patients with this type of cancer. This therapy may further be optionally combined with cimiprimab, pembrolizumab, and / or nivolumab.

[0264] For individuals with liver cancer (hepatocellular carcinoma) who have received sorafenib treatment

[0265] If a patient with liver cancer (hepatocellular carcinoma) has previously received sorafenib treatment, then P1801, or a combination of it with another molecule (such as P1101), can be used to treat that individual. This therapy may further be optionally combined with cimiprimab, nivolumab, pembrolizumab, and / or ipilimumab.

[0266] Additionally, P1801, or in combination with another molecule (e.g., P1101), can be used to treat adults and children 12 years of age and older with a type of colorectal cancer (colonic cancer) that has spread to other parts of the body (metastasis), exhibits high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR), and in individuals who have previously been treated with fluoropyrimidine, oxaliplatin, and irinotecan, and these drugs have been ineffective or no longer effective. This therapy may further be optionally combined with cimiprimab, pembrolizumab, and / or nivolumab.

[0267] x. Application route and dosage

[0268] P1801, or a combination of it with another molecule (e.g., P1101, nivolumab, and / or ipilimumab), can be formulated as an injection for intravenous (IV) use. In one embodiment, nivolumab (about 10 mg / mL) and ipilimumab (about 5 mg / mL) are an injection for intravenous (IV) use. In one embodiment, the amount of P1101 may be between about 50 and about 540 μg. Other non-limiting examples include six consecutive doses of 450 mcg P1101 (Q2W) together with 0.3 mg / kg P1801 (Q2W); six consecutive doses of 450 mcg P1101 (Q2W) together with 0.75 mg / kg P1801 (Q2W); six consecutive doses of 450 mcg P1101 (Q2W) together with 1.5 mg / kg P1801 (Q2W); six consecutive doses of 450 mcg P1101 (Q2W) together with 2.0 mg / kg P1801 (Q2W); or six consecutive doses of 450 mcg P1101 (Q2W) together with 3.0 mg / kg P1801 (Q2W). The treatment period can be during a treatment cycle of 28 days, 35 days, 42 days, 49 days, or 56 days.

[0269] Example

[0270] Example 1

[0271] Binding affinity and inhibition of P1801

[0272] This example aims to measure the binding affinity and inhibitory capacity of the P1801 antibody to the antigen. A direct binding ELISA was performed to measure binding specificity and cross-reactivity. Competitive ELISA and a cell-based reporter gene assay were performed to measure the inhibitory capacity of P1801 PD1 / PD-L1 versus PD1 / PD-L2.

[0273] All incubations should be performed at 25°C, except for antigen-coated culture plates at 4°C. Keep culture plates away from airflow and other temperature fluctuations, and cover them with a culture plate seal. During all washing steps, add 300 μL / well of wash buffer, then remove the solution from the culture plates by inverting them and wiping them on a paper towel. Repeat each washing step three times.

[0274] The antigen was diluted to an appropriate concentration with 1×DPBS at 50 μL / well and incubated overnight, followed by blocking with 1% BSA at 200 μL / well for 2 hours. Sample preparation and incubation: P1801 samples were diluted with 1% BSA, starting with a 2.5-fold serial dilution at 0.8 μg / mL for 8 spots. Two copies of freshly prepared serially diluted sample solution were then added to each well. The plate was sealed and incubated at 25°C without shaking for 1 hour. Secondary Ab incubation was performed by diluting the peroxidase-bound goat anti-human IgG Fe fragment-specific Ab to 1:10000 in 1% BSA, followed by adding 50 μL of freshly prepared diluted secondary Ab solution to each well. The plate was sealed and incubated at 25°C without shaking for 40 minutes. Color development was performed by adding 50 μL / well of TMB substrate solution, gently tapping the plate to ensure thorough mixing, and allowing it to stand for 2–5 minutes for color development. After incubation, terminate the reaction directly with 50 μL / well of stop solution. Read the OD within 10 minutes and protect from light. Ensure all wells are free of air bubbles.

[0275] Inhibition of PD-1 / PD-L1 and PD-L2 was measured by competitive binding ELISA.

[0276] For plating and blocking, the antigen was diluted to an appropriate concentration overnight with 1× DPBS at 50 μL / well and blocked for 2 hours with 1% BSA at 200 μL / well. For competitor P1801 sample preparation and incubation: using 1% BSA as dilution buffer, 10 consecutive 2-fold dilutions were performed starting at 5 μg / mL. Then, 50 μL of sample dilution was added to each appropriate well. For the binding agent biotin-hPD-1-mlgG Fe, it was diluted to a working solution of 0.25 μg / mL and then 50 μL was added to each well. The plate was incubated at 25°C with constant shaking (550 rpm) for 2 hours. Secondary Ab incubation was performed by diluting anti-streptavidin HRP Ab to 1:15000 in 1% BSA and then adding 50 μL of freshly prepared diluted secondary Ab solution to the wells of the plate. The plate was sealed and incubated at 25°C with constant shaking (550 rpm) for 40 minutes. Develop the color as follows: Add 50 μL / well of TMB substrate solution, gently tap the culture plate to ensure thorough mixing, and incubate in the dark for 30 minutes. After incubation, terminate the reaction directly with 50 μL / well of stop solution. Read the OD within 10 minutes and protect from light. Ensure all wells are free of air bubbles.

[0277] An analytical method for cell-based reporter gene assays was used to measure the inhibition of PD-1 / PD-L1 by the P1801 anti-PD1 antibody. Simply put, Jurkat cells stably expressing PD-1 (Jurkat / PD1) express a luciferase reporter gene driven by the NFAT response element (NFAT-RE) via TCR activation. However, although PD-1 binds to PD-L1 on Jurkat cells, this interaction blocks TCR-mediated luminescence. When Jurkat cells are activated by a TCR agonist and the PD-1 / PD-L1 interaction is disrupted by anti-PD1, luciferase (PD-1 / PD-L1) is induced. Figure 9 The greater the anti-PD1 blocking interaction, the more luciferase is induced. IC 50 It can be determined by the dose-response curve of the test sample.

[0278] Table 2 below shows the in vitro binding specificity of P1801.

[0279] Table 2

[0280]

[0281] The cross-reactivity of P1801 is reported in Table 3 below:

[0282] Table 3

[0283]

[0284] Assay system: Directly binds to ELISA

[0285]

[0286] Table 4 shows the inhibitory activity of P1801.

[0287] Table 4

[0288]

[0289] In short, P1801 has a 0.57 nM affinity for human PD-1 (EC1). 50 ) and 0.44 nM PD-1 affinity in cynomolgus monkeys (EC 50 No significant difference was observed in the binding affinity of P1801 to PD-1 between humans and cynomolgus monkeys. No cross-reactivity was observed between P1801 and mouse PD-1. Competitive interaction between P1801 and human PD-1 was observed in both competitive ELISA and cell-based assays for human PD-1 and human PD-L1 / PD-L2. P1801 binds to human PD-1 and cross-reacts with monkey PD-1, and the binding efficacy between human PD-1 and monkey PD-1 (ECG) was also observed. 50 No significant differences were observed. P1801 did not cross-react with mouse PD-1. P1801 inhibited the binding of PD-1 to PD-L1 and PD-L2 in competitive binding ELISA and cell-based reporter gene assays. P1801 responded to human and cynomolgus monkey PD-1, but not to mouse PD-1.

[0290] Example 2

[0291] Epitope combination of P1801

[0292] The epitope binding site of P1801 was compared with two existing commercially available anti-PD1 mAbs (pembrolizumab and nivolumab). Biomolecular interactions were determined using BLI (Biodesorption Ligation) technology. This technique is an optical analysis method that analyzes the interferogram of white light reflected from two surfaces: an internal reference layer and a protein layer deposited on the biosensor tip. Changes in the interferogram are induced by any variation in the number of molecules bound to the biosensor tip. In this case, interactions can be measured in real time, providing the ability to monitor binding specificity, association-to-dissociation ratio, concentration, fidelity, and accuracy. Furthermore, only molecules bound to or dissociated from the biosensor can alter the interferogram and produce a reaction spectrum on the BLI system. However, unbound molecules, changes in the refractive index of the surrounding medium, or changes in flow rate have no effect on the interferogram.

[0293] Here, the blocking effect of the purified anti-PD1 monoclonal antibody P1801 on the PD-1 epitope was tested using Octet BMIA. The purified PD1RB antibody was pre-incubated for 1 hour with PD-1 / His (10 μg / mL antibody; 0.336 μg / mL PD-1). An anti-human IgG (Fc) sensor was loaded with 3 μg / mL of various baseline antibodies. The association of the pre-formed antibody + antigen complex was measured and compared with the association of the antigen alone. High binding reaction indicated no epitope overlap, while lower reaction indicated epitope overlap. Baseline run for 30 seconds. The antibody loaded on the sensor was run at 4 μg / mL for 700 seconds. Quenching was performed for 480 seconds, and baseline run for 300 seconds. 10 μg / mL of blocking antibody associated with 0.336 μg / mL PD-1 for 600 seconds. Detailed results are shown in Figure 8(a) comparing nivolumab to P1801 and Figure 8(b) regarding pembrolizumab. If P1801 binds to the same epitope as nivolumab or pembrolizumab, then the lines will overlap, but they are not. Conversely, the two lines marked with arrows do not overlap with any other lines. This indicates non-overlapping binding on the target and represents partial overlap.

[0294] For both benchmark antibodies, P1801 showed a response of 0.0453 against nivolumab and 0.0426 nm against pembrolizumab. Therefore, the results indicate that P1801 is a partial blocker of PD1 compared to the two commercially available anti-PD1 mAb epitope binding sites, but the other characteristics exhibited by P1801 are comparable, which is surprising and unexpected.

[0295] Example 3

[0296] Combining dynamics

[0297] This example demonstrates the measurement of the binding affinity of the P1801 antibody to an antigen using a Biacore 8K. First, FcRn and C1q were immobilized onto a CM5 sensor chip. FcRn and C1q were immobilized at 25°C with HBS EP used as the run buffer. The sensor chip surfaces of flow cells 1 and 2 were activated for 200 seconds (10 μL / min) with a freshly mixed 50 mmol / L N-hydroxysuccinimide (NHS) and 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Subsequently, FcRn and C1q diluted in 30 mmol / L NaAC (pH 4.5) were injected into flow cell 2 to achieve binding of the appropriate reaction units, while flow cell 1 was set as a blank. Following the amine coupling reaction, any remaining active coupling sites on the chip surface were blocked by a 200-second injection of 1 mol / L ethanolamine hydrochloride. To measure the affinity of FcRn and C1q, measurements were performed at 25°C with HBS EP (pH 6.0) as the run buffer. Diluted P1801 antibody was injected onto the surfaces of flow cells 1 and 2 as the associative phase, followed by run buffer as the dissociation phase. Next, the affinity for human PD1 was measured. Measurements were performed at 25°C with HBS EP (pH 7.4) as the run buffer. Diluted P1801 antibody (2 μg / ml) was injected onto the surfaces as the capture phase, human PD1 protein was injected onto the surfaces as the associative phase, followed by run buffer as the dissociation phase. Finally, P1801 Fc binding was characterized by SPR, where the kinetic data for P1801 antibody to human PD1, FcRn, and C1q were obtained through affinity measurements. All data were processed using Biacore 8K evaluation software version 1.1. Buffer blank injections in flow cell 1 and each cycle were used as a dual reference for reaction unit subtraction.

[0298] The data is listed in Table 5 below.

[0299] Table 5

[0300]

[0301] In short, in both drug batches, P1801 binds to human PD-1 with an affinity (KD) of 2.50 and 3.13 nM, and P1801 binds to FcRn and C1q with an affinity (KD) comparable to that of pembrolizumab.

[0302] Example 4

[0303] Dynamic measurements of CD64

[0304] This example demonstrates a multi-cycle kinetic analysis of human PD1, cynomolgus monkey PD1, and human CD64 with anti-PD1 Ab. In this example, the anti-PD1 Ab ligand was captured on the surface of sensor chip protein A. The multi-cycle kinetic analysis of human PD1, cynomolgus monkey PD1, and human CD64 with anti-PD1 Ab was performed using a Biacore T200. The anti-PD1 Ab ligand was first diluted with 1×HBS-EP+ to obtain a final concentration of 3 μg / ml and captured by flow cell 2 of sensor chip protein A. The chip contains MabSelect SuRe ligand on its surface, allowing for targeted and specific binding to the Fc region of the antibody. The assay was performed using a Kinetic / Affinity wizard. The flow path was 2-1 when the ligand was injected and captured in flow cell 2, with flow cell 1 used as a reference. A series of analytes (human PD1, cynomolgus monkey PD1, and human CD64) were sequentially injected into the reference and ligand surfaces as associative phases, with transient dissociation phases obtained by injection of operating buffer. Subsequently, regeneration solution was injected as the regeneration phase. All obtained data were fitted to a 1:1 binding model using Biacore T200 evaluation software version 3. Blank injections of flow cell 1 and operating buffer were used as dual references for subtraction. The results are summarized in Table 6.

[0305] Table 6. Binding affinity of P1801 to CD64 (IgG receptor FcγRI) as determined by SPR

[0306]

[0307] In short, P1801 binds to human PD-1 with an affinity (KD) of 1.48 nM and to cynomolgus monkey PD-1 with an affinity (KD) of 1.62 nM. P1801 has a molecular weight of 2.96 × 10⁻⁶. -10 M-type human CD64 (IgG receptor FcγRI) affinity (KD).

[0308] Example 5

[0309] Internal therapeutic effects

[0310] This example demonstrates the in vivo therapeutic efficacy of compound P1801 in treating a HuCell MC38-hPDL1 model in female hPD-1 HuGEMM mice, as well as the effect of P1801 + mP1101. Each mouse was subcutaneously inoculated with 0.1 ml of HuCell MC38 tumor cells (1 × 10⁻⁶) in PBS in the right posterior ventral region. 6 (This is to allow the tumor to develop. When the tumor reaches approximately 65mm...) 3Treatment was initiated when the average tumor volume was reached. Treatment with the test compound P1801 significantly reduced the average tumor volume compared to the caustic agent group. Thirty mice participated in the study. All animals were randomly assigned to three different study groups. The average tumor size at randomization was approximately 65 mm. 3 The date of tumor randomization is marked as day 0. Morbidity and mortality were assessed daily after tumor cell inoculation. During routine monitoring, the animals were examined for any effects of tumor growth and treatment on behavior, such as activity level, food and water consumption, weight gain / loss (measured twice weekly after randomization), and any other abnormalities. Individual mouse mortality and observed clinical signs were recorded. Tumor volume was measured twice weekly in two dimensions using calipers, and the volume was expressed in mm using the following formula. 3 The formula is: V = (L×W×W) / 2, where V is the tumor volume, L is the tumor length (longest tumor size), and W is the tumor width (longest tumor size perpendicular to L). Tumor weight was measured at the end of the study. Drug administration and tumor and body weight measurements were performed in a laminar flow cabinet.

[0311] On day 23 post-vaccination, the MTV in the isotype control group reached 3489.56 mm. 3 Furthermore, the MTV in the P1801 12mpk treatment group and the nivolumab 12mpk treatment group reached 39.39 mm. 3 and 236.69 mm 3 Compared with the isotype control group, the test substance P1801 showed significant tumor suppression in the subcutaneous HuCell MC38-hPDL1 model in female hPD-1 HuGEMM mice (TGI: 98.87%) (p<0.001). During follow-up, complete response (CR) was observed in 6 mice in the P1801 treatment group and in 8 mice in the nivolumab treatment group. In terms of survival, the median survival time of P1801 treatment exceeded 50 days, significantly longer than that of the isotype control group (p = 0.002). The median survival time of the nivolumab 12mpk positive control group also exceeded 50 days, significantly longer than that of the isotype control group (p<0.001). For example, one administration regimen can be found in… Figure 6 (a) and Figure 6 (b) in.

[0312] The overview of tumor growth inhibition is shown in Table 7 below:

[0313] Table 7

[0314]

[0315] Data showed that, compared with the isotype control group, P1801 inhibited the growth of MC38-hPD-L1 tumors in hPD-1 mice at a dose of 12 mg / kg BIW×3. Furthermore, compared with the isotype control group, P1801 prolonged the survival of tumor-bearing hPD-1 mice. The combination of mP1101 and P1801 also demonstrated efficacy.

[0316] Example 6

[0317] IL2-induced

[0318] This example includes the results of a whole blood cytokine response assay for P1801. This assay observed that P1801 enhances IL-2 production in SEB1-stimulated whole blood in a dose-dependent manner. Heparinized blood from healthy donors was pre-cultured with P1801 and subsequently treated with SEB to induce IL-2 release. IL-2 induction can further modulate and / or activate T-cell responses, thereby inhibiting and / or treating cancer.

[0319] Two days after treatment, the supernatant was collected, and IL-2 was quantified by ELISA. Whole blood was diluted tenfold with culture medium and pre-incubated for 60 minutes in a 96-well plate at 37°C, 5% CO2, with or without the test substance and NC (0.04, 0.2, 1, 5, and 25 μg / mL). Except for the control wells (without SEB induction), whole blood cells were subsequently stimulated with SEB (0.1 μg / mL) and incubated at 37°C, 5% CO2 for 48 hours. After incubation, the supernatant was collected by centrifugation at 500xg for 5 minutes, aliquoted, and stored at -30°C until ELISA analysis. IL-2 was quantified by ELISA following the manufacturer's instructions. In short, the standards and supernatant were serially diluted with correction diluent RD5-5. Add 100 μL of assay diluent RD1W to each well, followed by 100 μL of blank standard (31.3 to 2000 pg / mL) and diluted supernatant (2-fold, 4-fold, 8-fold, and 16-fold dilutions) to the designated wells, and continue for two hours. Wash the wells three times with diluted wash buffer, then add 200 μL of human IL-2 conjugate to each well, and continue for two hours. Repeat the washing steps and add 200 μL of substrate solution to each well, continuing for 10 to 20 minutes. Finally, add 50 μL of stop solution and measure the optical density (OD) using a microplate reader set to 450 and 570 nm². Analyze the data using Gen5™ software as follows: (A) Average the repeated readings of blank, standard, and supernatant, then subtract the average OD of the blank to obtain the result. OD. (B) Through The O.D. generates a standard curve and generates a four-parameter logistic (4-PL) curve fit. The IL-2 concentration of all supernatants read from the standard curve must be multiplied by the dilution factor. (C) Acceptance criteria a. If the O.D. is below the lowest standard (LLOQ3), then the IL-2 value will be denoted by the symbol "<LLOQ" and excluded from subsequent calculations. If the RPD 4 calculated based on the IL-2 values of the two specified dilutions is higher than 20%, then the concentration of the lower dilution will be reported. Conversely, the average of the concentrations of the two dilutions will be reported.

[0320] The resulting pharmacodynamic results are summarized in Table 8 below:

[0321] Table 8

[0322] IL-2 induced by SEB in human whole blood

[0323]

[0324]

[0325] The results showed that compared with the IgG4 isotype control, at 0.2, 1, 5 and 25 μg / mL, P1801 induced a significant difference in IL-2 production in human whole blood. 2. At 0.04, 1, 5 and 25 μg / mL, there was no significant difference in IL-2 production induced by P1801 and nivolumab (P value > 0.05).

[0326] For each dose level, N = 8.

[0327] IL-2 was not detected in whole blood without SEB stimulation. At concentrations of 0.2, 1, 5 and 25 μg / mL, there were statistically significant differences in IL-2 production between P1801 and human IgG4 (p values were 0.019, 0.001, 0.001 and 0.015 respectively). At concentrations of 0.04, 1, 5 and 25 μg / mL, there was no statistically significant difference in IL-2 production between P1801 and nivolumab (p value > 0.05). In the P1801 treatment group, the specific fold change relative to IgG4 was up to 1.49-fold and was comparable to nivolumab. P1801 was able to enhance IL-2 production in SEB-stimulated whole blood in a dose-dependent manner.

[0328] Example 7

[0329] This study evaluated pharmacokinetics, anti-drug antibody (ADA), receptor occupancy (RO), and IL-2 release in cynomolgus macaques following a single intravenous infusion of P1801 at three dose levels. All cynomolgus macaques assigned to the study were identified as healthy animals by routine blood tests prior to administration. Actual body weight and administration details were measured after the single intravenous injection. Weekly body weight records were maintained. Minor changes in body weight attributable to blood collection were observed during the period from administration to sample collection, and no abnormalities were observed. Blood was collected from all surviving animals before administration (days -7 and -1), 24 hours after administration, and at 168 hours (day 7), 672 hours (day 28), and 1334 hours (day 56) after administration for IL-2 release analysis. IL-2 release from monkey leukocytes was tested in untreated monkeys treated in vitro with 0.04, 0.2, 1, 5, or 25 μg / mL P1801, nivolumab, or isotype (IgG4) (before administration, -7 days). In the SEB stimulation study, P1801 and nivolumab treatments promoted IL-2 release in a dose-dependent manner, while IgG4 treatment induced only a slight increase in IL-2 release, which did not increase with increasing dose.

[0330] IL-2 release from monkey leukocytes was measured in monkeys treated with a single intravenous infusion of 1, 5, or 20 mg / kg P1801. Whole blood was collected from monkeys at five time points during the pharmacokinetic studies (day -1, day 1, day 7, day 28, and day 56). Time-dose-dependent IL-2 release was observed in the 5 and 20 mg / kg groups, but no significant dose-dependent IL-2 release from P1801 was observed. An overview is provided in Tables 9 and 10 below.

[0331] Table 9

[0332]

[0333]

[0334] In short, Table 9 above shows that P1801 promotes significant IL-2 production in monkey PBMCs in a dose-dependent manner, while the IgG4 isotype control causes a slight increase in IL-2 that does not increase with increasing dose. For each dose level, N=12.

[0335] Table 10

[0336]

[0337] This example demonstrates that P1801 promotes significant IL-2 production in whole blood of monkeys in a time-dependent manner. No dose dependence was observed when P1801 was administered at 1, 5, or 20 mg / kg. For each dose level, N=4 (2 males and 2 females); 1 p-value relative to day -1 (ANOVA / Kruskal-Wallis & Wilcoxon rank sum test on group means after Bartlett test).

[0338] Example 8

[0339] In vitro PD-1 receptor occupancy (RO).

[0340] Newly isolated human PBMCs were stimulated for 2 days with anti-CD3 / CD28-coated Dynabeads to induce PD-1 expression, and then cultured with a specified concentration of P1801. P1801-bound PD-1+ T cells were detected using a fluorophore-bound secondary antibody (anti-human IgG4 Fc antibody) and analyzed by flow cytometry. Human PBMCs were isolated from heparinized blood of healthy donors. Mouse anti-human IgG4 Fc antibody containing the fluorophore R-phycoerythrin (PE) and mouse anti-human CD3 antibody containing the fluorophore allophycocyanin (APC) were used. A negative control was a recombinant human IgG4 isotype control antibody purified by Ultra-LEAF™. The human T-cell activator Dynabeads (trademark) CD3 / CD28 was used. Human PBMCs were isolated from heparinized whole blood of healthy donors no more than 6 hours after collection using Ficoll-Paque (trademark) PLUS. Human PBMCs were stimulated for 2 days at 37°C and 5% CO2 incubator to express PD-1 using Dynabeads (trademark) human T-cell activators CD3 / CD28. After two days of activation, PBMCs were suspended and Dynabeads were removed using a magnetic rack (DynaMag) (trademark). PBMCs were incubated for 2 × 10⁶ cells in 100 μL staining buffer at room temperature in the dark, with or without P1801 (0.01, 0.1, and 1 μg / mL), a comparison (0.01, 0.1, and 1 μg / mL), and a negative control (1 μg / mL). 5 Incubate 1 PBMC for 30 minutes. Wash the PBMCs twice with staining buffer before staining. Dilute anti-hCD3 and anti-hIgG4 Fc antibodies in staining buffer and add 100 μL of the diluted antibody to a tube containing the PBMCs. Incubate in the dark at room temperature for 20 minutes.

[0341] PBMCs were washed twice with 1 mL staining buffer to remove unbound antibodies. The cell pellet was resuspended in 350 μL staining buffer and analyzed by flow cytometry as soon as possible. Data were analyzed using FCS Express software as follows: (A) CD3-positive regions of the FSC / CD3 dot matrix were circled for further analysis of PD-1 expression, including the percentage of PD-1+ cells (PD-1+%) and the geometric mean fluorescence intensity (Geo MFI) of PD-1 on all CD3+ T cells. (B) PD-1+% was defined as the percentage of CD3+ T cells bound to the anti-PD-1 antibody and was calculated by subtracting the untreated group from the treated group using the Overton method. (C) Geo MFI was defined as the geometric mean fluorescence intensity of the PE channel and represents the degree of binding of the anti-PD-1 antibody to PD-1 on the surface of all CD3+ T cells. Data in the summary table are presented as mean ± SE3 for ten donors across eight independent experiments, and P1801 and comparisons were analyzed by single-factor ANOVA4. Results are summarized in Table 11 below.

[0342] Table 11

[0343] P1801 receptor occupancy in human PBMCs

[0344]

[0345]

[0346] The results showed that the percentage of P1801 bound to human PBMCs was dose-dependent. At 0.1 and 1 μg / mL, the percentage of PD-1 positive CD3+ T cells was not significantly different between P1801 and nivolumab. Furthermore, the PD-1 binding activity of P1801 showed a dose-dependent effect. At intermediate and high concentrations of 0.1 and 1 μg / mL, respectively, there was no statistically significant difference in PD-1+% between P1801 and nivolumab (p>0.05). At a high concentration of 1 μg / mL, there was no statistically significant difference in Geo MFI between P1801 and nivolumab (p>0.05).

[0347] Example 9

[0348] In vivo therapeutic efficacy of Renca subcutaneous syngeneic renal cell carcinoma model in female BALB / c mice

[0349] To evaluate the antitumor efficacy of the combination of anti-PD1 antibody and P1101 in a mouse model, two mouse formulations of the molecules were used: mP1101, in which mouse interferon was used instead of the human formulation, and the known mouse anti-PD1 mAb RMP 1-14, which was used as the anti-PD1 mAb. The study design included: randomization to the study group; daily administration, 7 days a week for 3 weeks; a post-treatment observation period based on the implantation site; and subcutaneous or in situ administration. During the administration and observation periods, animals were examined daily, 5 days a week; for subcutaneous implantation, body weight and tumor volume were measured twice weekly.

[0350] Table 12 shows the comparison with Figures 4(a) to 4(c) A similar design overview illustrates the administration of the combination and the frequency of various molecules. In Table 12 below, Q3D represents administration every 3 days. ×6 indicates 6 administrations. SC represents subcutaneous administration. IP represents intraperitoneal injection. ROA represents the route of administration.

[0351] Table 12

[0352]

[0353]

[0354] method

[0355] Renca (CL-00796) cells were maintained in vitro at 37°C and in an atmosphere of 5% CO2 using DMEM medium supplemented with 10% fetal bovine serum. Cells in the exponential growth phase were collected and quantified using a cell counter, followed by tumor inoculation. For tumor inoculation, 0.1 ml of Renca tumor cells (1 × 10⁻⁶) in PBS was injected into the right lower ventral region of each mouse. 6 Subcutaneous inoculation was used to develop tumors. The average tumor size reached approximately 70 mm. 3 Randomization began at that time. Fifty-six mice were selected for the study. All animals were randomly assigned to seven study groups of eight mice each. Randomization was performed using a matched distribution / stratified method (StudyDirector software, version 3.1.399.19). The randomization day was designated as day 0. Treatment began on the same day as randomization (day 1), according to the study design.

[0356] Observation results and data collection

[0357] Following tumor cell inoculation, morbidity and mortality were assessed daily in the animals. During routine monitoring, the animals were examined for tumor growth and any effects of treatment on behavior, such as activity level, food and water consumption, weight gain / loss (measured twice weekly after randomization), eye abnormalities / hair tangles, and any other abnormalities. Individual animal mortality and observed clinical signs were documented in detail. Tumor volume was measured twice weekly in two dimensions using calipers after randomization, and the volume was expressed in mm using the following formula. 3 The expression is: "V = (L×W×W) / 2", where V is the tumor volume, L is the tumor length (longest tumor size), and W is the tumor width (longest tumor size perpendicular to L). Drug administration and tumor and body weight measurements were performed in a laminar flow hood. Body weight and tumor volume were also measured using StudyDirector software.

[0358] Treatment lasted 33 weeks. The study was terminated on day 33. Tumors from euthanized mice were not collected prior to study termination. No samples were taken from any dead mice found.

[0359] Statistical analysis

[0360] First, we perform statistical analysis using the Bartlett test to test for homogeneity of variance. Second, if the p-value of the Bartlett test is greater than 0.05, it indicates insufficient evidence to reject the null hypothesis of homogeneity of variance, which is one of the hypotheses of ANOVA. Therefore, we perform ANOVA to test whether any pair of groups has different group means, and perform a post-hoc Tukey HSD on all paired comparisons, or a Dunnett's t-test on each treatment and one control. If the p-value of the Bartlett test is less than or equal to 0.05, it indicates some evidence that the variances may be unequal or that the data do not follow a normal distribution. Next, we perform the Kruskal-Wallis test to test whether any pair of groups has different group medians, and perform a post-hoc Conover's non-parametric all-pairs comparison test on all paired comparisons, or a Conover non-parametric many-to-one comparison test on each treatment and one control. Both tests use single-step p-adjustment. In addition, pairwise comparisons are performed without multiple comparison correction, and the nominal / uncorrected p-values ​​are reported directly based on Welch's t-test or the Mann-Whitney U test. The criteria for choosing the Welch t-test or the Mann-Whitney U test are the same as for two-group comparisons.

[0361] All statistical analyses were performed in the R language and environment (version 3.6.0) for statistical computation and graphics. Unless otherwise specified, all tests were two-tailed, and p-values ​​<0.05 were considered statistically significant. Survival analyses applied the Kaplan-Meier model with the log-rank method.

[0362] result

[0363] First, body weight growth curves were measured. Results of body weight and changes in body weight at different time points were presented. Tumor growth curves (mean tumor volume over time) for different groups are shown below. Figure 10 Detailed measurements and statistics are shown in Table 13.

[0364] Table 13

[0365]

[0366] In this case, the mean tumor volume in Group 1 reached 3486.36 mm on day 22 after the start of treatment. 3 Group 2 was treated with anti-PD-1 at 12 mg / kg, with a TGI of 1.59% on day 22. Group 3 was treated with mP1101 at 0.1 mg / kg, with a TGI of 70.70% on day 22. Group 4 was treated with mP1101 at 0.1 mg / kg Q3D×3 and anti-PD-1 at 12 mg / kg Q3D×6, with a TGI of 64.51% on day 22. Group 5 was treated with mP1101 at 0.1 mg / kg Q3D×6 and anti-PD-1 at 12 mg / kg Q3D×6, with a TGI of 79.59% on day 22. Group 6 was treated with mP1101 at 0.1 mg / kg Q3D×3 followed by anti-PD-1 at 12 mg / kg Q3D×6, with a TGI of 63.67% on day 22. Group 7 was first treated with mP1101 0.1 mg / kg Q3D×6, followed by anti-PD1 12 mg / kg Q3D×6. The TGI value on day 22 was 76.40%.

[0367] The median survival time (MST) of each group was also studied. Mice that reached the humane endpoint were euthanized. Treatment options included: median control, anti-PD1 12 mg / kg, mP1101 0.1 mg / kg, mP1101 0.1 mg / kg Q3D×3, anti-PD1 12 mg / kg Q3D×6, mP1101 0.1 mg / kg Q3D×6, anti-PD1 12 mg / kg Q3D×6, mP1101 0.1 mg / kg Q3D×3 followed by anti-PD1 12 mg / kg Q3D×6, and mP1101 0.1 mg / kg Q3D×6 followed by anti-PD1 12 mg / kg. The MSTs for mg / kg Q3D×6 were 23.00 days, 23.00 days (relative to the carrier control group, P>0.05), 31.50 days (relative to the carrier control group, P<0.01), 29.00 days (relative to the carrier control group, P<0.01), 16.50 days (relative to the carrier control group, P>0.05), 30.00 days (relative to the carrier control group, P<0.001), and 33.00 days (relative to the carrier control group, P<0.01).

[0368] In short, in this example, the therapeutic efficacy of the test agent mP1101 was evaluated in a rodent Renca model in female BALB / c mice. Compared with the mediator group, mP1101 alone (Group 3) and in combination with anti-PD1 at 12 mg / kg (Groups 4, 5, 6, and 7) showed significant antitumor efficacy (P < 0.001). Furthermore, mP1101 alone (Group 3) and in combination with anti-PD1 at 12 mg / kg (Groups 4, 6, and 7) prolonged the survival of mice.

[0369] Example 10

[0370] Therapeutic effects

[0371] To evaluate the antitumor efficacy of the anti-PD1 mAb P1801, mammalian individuals were treated with an amount of P1801 that effectively reduced, inhibited, and / or treated CRC, RCC, and / or melanoma.

[0372] Each individual with RCC or melanoma was treated. Treatment with P1801 resulted in a reduction in mean tumor volume compared to the mediator control group. Thirty individuals were selected for the study. All individuals were randomly assigned to three different study groups. The mean tumor size at the time of initial randomization was approximately 65 mm. 3The date of tumor randomization is marked as day 0. All individuals are examined daily. During routine monitoring, individuals are examined for any effects of tumor growth and treatment on behavior, such as activity level, food and water consumption, weight gain / loss, and any other abnormalities. Mortality and observed clinical signs are recorded for each individual. Tumor volume is measured twice weekly, and the volume is expressed in mm using the following formula. 3 The formula is: V = (L×W×W) / 2, where V is the tumor volume, L is the tumor length (the longest tumor size), and W is the tumor width (the longest tumor size perpendicular to L). Tumor weight was measured at the end of the study.

[0373] Application instructions can be found at Figure 12 In appropriate cases, individuals may show a reduction in the size of melanomas or RCCs.

[0374] Example 11

[0375] To evaluate the antitumor efficacy of anti-PD1 P1801 mAb alone, P1101 alone (a human-formed interferon), and / or the combination of anti-PD1 mAb P1801 + P1101 in mammalian individuals, human forms of both molecules were used: P1101 and P1801 (humanized forms of human interferon). The study design included randomization to the study group; daily administration, 7 days a week for 3 weeks; a post-treatment observation period based on the implantation site; and subcutaneous or in situ administration. During the administration and observation period, human individuals were examined daily, 5 days a week; for subcutaneous implantation, body weight and tumor volume were measured twice weekly.

[0376] Table 13 presents an overview of the experimental design, and Figures 4(a) to 4(c) The dosage of the combination and the frequency of each molecule are shown. In Table 14 below, Q3D represents administration every 3 days. ×6 means 6 times. SC represents subcutaneous administration. IP represents intraperitoneal injection.

[0377] Table 14

[0378]

[0379] Results showed that in human individuals treated with RCC, CRC, and / or melanoma, all of the following reduced tumor size in RCC, CRC, and / or melanoma: P1801 alone, P1101 alone, and combination therapy with P1801 and P1101.

[0380] Example 12

[0381] Continuous dose escalation in clinical trials

[0382] To examine the toxicity / tolerability levels of P1801 + P1101 in humans, a Phase 1 trial was conducted in humans. The starting dose of P1801 was determined based on preclinical pharmacological and toxicological data. As shown in the example above, a NOAEL of 200 mg / kg was reported in a preclinical toxicity study in cynomolgus monkeys. Taking into account a conversion factor of 3.1 (monkey to human) and a safety margin of 6, the maximum recommended starting dose of P1801 was 10.75 mg / kg (200 mg / kg divided by 3.1, then divided by 6). For more stringent safety considerations, 0.3 mg / kg was chosen as the starting dose for this example.

[0383] Initially, individuals meeting the criteria (e.g., age >18, having solid tumors such as renal cell carcinoma (RCC), melanoma, hepatocellular carcinoma (HCC), and / or CRC, with some patients potentially at an advanced stage) will be screened. Individuals will also have a life expectancy of 3 months or more, normal thyroid function, and be negative for HBV, HCV, and HIV. In groups 1 through 5, individuals with advanced solid tumors (particularly including renal cell carcinoma (RCC), melanoma, hepatocellular carcinoma (HCC), and colorectal cancer (CRC)) will be treated in groups with six doses of P1101 at 450 mcg every 28 days during each 28-day treatment cycle, followed by P1801 every 22 days at the predetermined dose level.

[0384] For dose escalation, five different groups were used, such as Figure 12 As shown in the diagram. Once the effective dose is determined, group expansion is performed, as... Figure 13 As shown in the figure. PD-L1 expression was measured in all individuals, but only individuals with positive PD-L1 expression were selected into another group, as shown in the figure. Figure 14 As shown in the diagram. Note that in this example, P1101 and P1801 are administered sequentially and are not interchangeable. The results of this example show that the indicated effective dose is effective in treating CRC / HCC / RCC / melanoma.

[0385] Example 13

[0386] Complementary bit determination

[0387] The structure and 3D model of P1801 / PD-1 were determined using high-resolution cryo-electron microscopy, thus obtaining the atomic-resolution structure of P1801 / PD-1, revealing the spatial arrangement of amino acids in the 3D structure and identifying complementary sites.

[0388] To determine complementary sites, a grid-based selection was performed on the PD1-AcroBio sample, yielding the following dataset. A dataset of samples PD1-AcroBio + PS00066 + PS00067 was collected. The dataset was processed in cryoSPARC 4.4. Micrographs with poorly fitted contrast transfer functions (CTF < 4 A) were removed, and 5,306 micrographs were selected for further processing. Particle selection was performed based on a template, yielding approximately 4 million particles with a box size of 320 pixels (approximately 265 A). For initial cleaning, two rounds of 2D classification and selected 2D classes were performed, resulting in approximately 1.5 million particles. The 2D class averages show discrete and well-defined particles with different views and visible secondary structure features. The selected particles were first used in a de novo reconstruction round, followed by four heterogeneous refinements using three decoys, and one non-uniform refinement. The final 128 K-particle generation yielded the highest resolution map, which was then selected for further refinement using CTF global refinement, followed by local resolution refinement and reference-based motion correction. The resolution based on the gold-standard Fourier shell correlation estimate of 0.143 was 2.9 A (…). Figure 16-17 The half-map was further refined using the DeepEMhancer Python package implemented in cryoSPARC. The models for PD1 (α-sheet) and the two FABs were docked to usable densities using ChimeraX. The variable regions of the FABs were then mutated according to the customer-provided sequences PS00066 and PS00067. The constant regions are not shown. The resulting model underwent multiple rounds of manual reconstruction (using COOT) and computer-based refinement (Phenix). Glycans were manually constructed at positions ASN 49 and ASN 58. The final model was validated against the half-map, and its quality was assessed using MolProbability. The obtained structures provide the location of the protein-protein interface between PD1 and PS00066 / PS00067.

[0389] Sample and grid preparation for Cryo-EM. PD1-AcroBio + PS00066 + PS00067 was prepared by mixing sample PD1-AcroBio, sample PS00066, and sample PS00067 in a 1:1:1 molar ratio and incubating at room temperature for 30 minutes, yielding a final protein concentration of 0.18 mg / mL for the complex. The 1:1:1 molar ratio of sample PD1-AcroBio, sample PS00066, and sample PS00067 was diluted 17.5-fold in DPBS (provided by the customer) to obtain a final sample concentration of 0.01 mg / mL prior to grid preparation and imaging. A FEI Vitrobot instrument or manual plunger was used during conventional immersion freezing. In short, a 3 μl drop of sample suspension was coated onto the EM grid, which had been plasma-cleaned using GatanSolarus (Pleasanton, California). After wiping the sample with filter paper, the grid was immersed in liquid ethane for freezing. The grid is stored in liquid nitrogen until it is transferred to a transmission electron microscope for imaging.

[0390] Cryo-EM imaging was performed using a FEI Titan Krios microscope. Electron microscopy was performed using a FEI Titan Krios (Hillsboro, Oregon) transmission electron microscope operating at 300 kV, equipped with a Gatan Quantum 967 LS imaging filter, a Volta phase plate, and a Gatan K3 direct detection camera. Glassy ice grids were clamped into sample holders, transferred to a cassette, and then to the Krios automated feeder, all while maintaining the grids at a cryogenic temperature (below -170°C). Automated data collection was performed using Leginon software, where high-magnification films were obtained by selecting targets at lower magnifications.

[0391] Preprocessing and 2D classification analysis (cryoSPARC, Relion). Dose-weighted cinematic frame alignment was performed using MotionCor2 or full-frame or patch motion correction in cryoSPARC (Punjani et al., 2017) to account for platform drift and fasciculation. The contrast transfer function for each micrograph was estimated using CTFfind4, gCTF, or Patch CTF in cryoSPARC (Rohou et al., 2015; Zhang et al., 2016). Individual particles were selected using an automated picking scheme and extracted into a particle stack in Relion or cryoSPARC. The particles were then subjected to reference-free 2D alignment and classification in Relion or cryoSPARC.

[0392] CryoSPARC (Cryo-EM Single Particle Analysis Reconstruction and Classification) simplifies cryo-EM data processing, providing capabilities such as particle selection, 2D classification, 3D reconstruction, and refinement. First, the mesh is screened and the data is compiled. Based on the data generated from cryoSPARC 4.4 micrographs, a spectrum is produced. Figure 16 Images generated for the final 3D reconstruction of PD1-AcroBio + PS 00066 + PS00067 using ChimeraX at a nominal resolution of 2.9 Å. The generated atlas with the highest resolution was selected for further refinement using global CTF refinement, followed by local resolution refinement and reference-based motion correction. The resolution was 2.9 Å based on the gold standard Fourier shell correlation estimate of 0.143. The atlas was further refined using the DeepEMhancer Python package implemented in cryoSPARC. The models used for PD1 (α-fold) and the two FABs were docked to the available density using ChimeraX. Figure 17 The final 2.9A map generated using ChimeraX was colored using local resolution values ​​calculated with cryoSPARC 4.4. The variable region of the FAB was then mutated according to the customer-provided sequences of PS00066 and PS00067. The resulting model underwent multiple rounds of manual reconstruction (using COOT) and computer-based refinement (Phenix). The final model was validated against the map, and its quality was assessed using MolProbity. The obtained structure provides the location of the protein-protein interface between PD1 and PS00066 / PS00067. The model illustrates the hydrophobic and electrostatic interactions between PD1 and PS00067. Protein-protein contacts appear to be primarily driven by hydrophobic interactions, with a cluster of aliphatic and aromatic residues, such as Tyr 32, Pro 33, His 52, Tyr 54 (I:PS00067), Tyr 95 (M:PS00067), and Pro 39, Ala 40, Leu 41, Leu 42, Val 43, Thr 45, Thr 145 (PD1). Additionally, Arg 143 and Glu 141 (PD1) can form hydrogen bonds with Glu 99 (I:PS00067) and Tyr 101 (M:PS00067) / His 104 (I:PS00067), respectively.

[0393] Figure 18 Image of the structure of the P1801 / PD1 complex as resolved by cryo-electron microscopy. Figure 19Images of the structure of the P1801 / PL1 / PD1 complex, as resolved by cryo-electron microscopy, show the overlap between P1801 / PD1 and PD-L1 / PD1. Figure 20 The image shows the epitope location for epitope P1801, illustrating the relationship with heavy chains T59, S60, E61, S62, and V64. P83, E84 D85, R86, L128 And A129, as well as the light chain S87 and P130 K131 and A132 The combination of, in This indicates that the table positions are shared. Figure 21 Images showing the epitope localization of PD-L1 epitopes, illustrating the relationship with V64. , N66, Y68, S73, N74, Q75, T76, D77, K78, E84 G124, I126, L128 P130 K131 A132 The combination of I134 and E136, where This indicates that the table positions are shared. Figure 22 The model of P1801 CDR at the PD-1 interface.

[0394] Figure 23 This is a graph showing the binding of epitopes to anti-PD-1 antibodies. Figure 23 The study demonstrated that all antibodies could bind strongly to PD-1 in the presence of P1801, indicating their different epitopes. ISIS-clone19 was selected as the non-competitive Fa. Peresolimab is a humanized immunoglobulin G1 monoclonal antibody that stimulates human PD-1.

[0395] In one embodiment, the P1801 variable region heavy chain sequence of the CDR is defined as follows:

[0396] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATITGGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYLNYFDYWGQGTLVTVSS (SEQ ID No: 5).

[0397] The VHCDR1 sequence SSYDMS (SEQ ID NO: 18); the VHCDR2 sequence TITGGGSYTYYPDSVKG (SEQ ID NO: 10); and the VHCDR3 sequence PYLNYFDY (SEQ ID NO: 11) are shown.

[0398] The P1801 variable region light chain sequence of CDR was defined as follows:

[0399] EIVLTQSPATLSLSPGERATLSCRASQSISNNLHWYQQKPGQAPRLLIYYASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSNSWPLTFGQGTKLEIK (SEQ ID No: 6).

[0400] The VLCDR1 sequence RASQSISNNLH (SEQ ID NO: 12); the VLCDR2 sequence YASQSIS (SEQ ID NO: 13); and the VLCDR3 sequence QQSNSWPLT (SEQ ID NO: 14) are shown.

[0401] In another embodiment, the P1801 variable region heavy chain sequence of the CDR is defined as follows:

[0402] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATITGGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYLNYFDYWGQGTLVTVSS (SEQ ID No: 5).

[0403] The following sequences were shown: VHCDR1 sequence GFTFSSYDMS (SEQ ID NO: 19); VHCDR2 sequence TITGGGSYTYYPDSVKG (SEQ ID NO: 10); and VHCDR3 sequence ASPYLNYFDY (SEQ ID NO: 20). The light chain sequence of the P1801 variable region of the CDR was defined as follows.

[0404] EIVLTQSPATLSLSPGERATLSCRASQSISNNLHWYQQKPGQAPRLLIYYASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSNSWPLTFGQGTKLEIK (SEQ ID No: 6).

[0405] The VLCDR1 sequence RASQSISNNLH (SEQ ID NO: 12); the VLCDR2 sequence YASQSIS (SEQ ID NO: 13); and the VLCDR3 sequence QQSNSWPLT (SEQ ID NO: 14) are shown.

[0406] In one embodiment, the P1801 variable region heavy chain sequence that defines the CDR showing the complementary bit is defined as follows:

[0407] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATITGGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYLNYFDYWGQGTLVTVSS (SEQ ID No: 5).

[0408] The VHCDR1 sequence G was shown. F TF SSY DMS (SEQ ID NO: 19) can also be used for Gly Phe Thr Phe Ser Ser Tyr Asp Met Ser and numbered Gly1 Phe2 Thr3 Phe4 Ser5 Ser6 Tyr7 Asp8 Met9 Ser10. The VHCDR1 sequence has a total of [number] complementary bits across all complementary bits. Phe2Ser5Ser6 and Tyr7Therefore, Gly1, Thr3, Phe4, Asp8, Met9, and Ser10 can individually substitute for any amino acid residue. For example, Gly1 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Thr3 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val; Phe4 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val. l; Asp8 can independently replace Ala, Arg, Asn, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Met9 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and Ser10 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val.

[0409] VHCDR2 sequence TI TGGGSY TYYPDSVKG (SEQ ID NO: 10) can be Thr Ile Thr Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly and numbered Thr11 Ile12 Thr13 Gly14 Gly15 Gly16 Ser17 Tyr18 Thr19 Tyr20 Tyr21 Pro22 Asp23 Ser24 Val25 Lys26 Gly27. The VHCDR2 sequence has a total of [number missing] complementary bits across all complementary positions. Thr13 Gly14 Gly15 Gly16 Ser17 and Tyr18Therefore, Thr11, Ile12, Thr19, Tyr20, Tyr21, Pro22, Asp23, Ser24, Val25, Lys26, and Gly27 can individually substitute any amino acid residue. For example, Thr11 can independently substitute Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val; Ile12 can independently substitute Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Thr19 can independently substitute Ala, Arg, Asn, Asx, Cys, Glu, Gln, Glx, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and Thr19 can independently substitute Ala, Arg, Asx ... n, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val; Tyr20 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val; Tyr21 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx. Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val; Pro22 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val; Asp23 can independently replace Ala, Arg, Asn, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pr o, Ser, Thr, Trp, Tyr, or Val; Ser24 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val; Val25 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;Lys26 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr; and Gly27 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.

[0410] VHCDR3 sequence A S P YLN YF DY (SEQ ID NO: 20) could be Ala Ser Pro Tyr Leu Asn Tyr Phe Asp Tyr And the number is Ala30 Ser31 Pro32 Tyr33 Leu34 Asn35 Tyr36 Phe37 Asp38 Tyr39 The VHCDR3 sequence has a total of [number] complementary bits across all complementary bits. Ser31, Tyr33, Leu34, Asn35, Asp38 and Tyr39 Therefore, Ala30, Pro32, Tyr36, and Phe37 can individually substitute any amino acid residue. For example, Ala30 can independently substitute Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Pro32 can independently substitute Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val. l; Tyr36 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val; and Phe37 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val.

[0411] In another embodiment, the P1801 variable region heavy chain substitution sequence indicating the complementary position of the CDR is defined as follows:

[0412] EVQLVESGGGLVQPGGSLRLSCAASGFTFSYDMSWVRQAPGKGLEWVATITGGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYLNYFDYWGQGTLVTVSS (SEQ ID No:22)

[0413] The VHCDR1 substitution sequence G is shown. F TF SY DMS (SEQ ID NO: 21), can also be written as Gly Phe ThrPhe Ser Tyr Asp Met Ser and numbered Gly1 Phe2 Thr3 Phe4 Ser5 Tyr6 Asp7 Met8 Ser9. The VHCDR1 sequence has a total of [number] complementary bits across all complementary bits. Phe2, Ser5 and Tyr6 Therefore, Gly1, Thr3, Phe4, Asp8, Met9, and Ser10 can individually substitute for any amino acid residue. For example, Gly1 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Thr3 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val; Phe4 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val. l; Asp7 can independently replace Ala, Arg, Asn, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Met8 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and Ser9 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val.

[0414] VHCDR2 sequence TI TGGGSY TYYPDSVKG (SEQ ID NO: 10) can be Thr Ile Thr Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly and numbered Thr11 Ile12 Thr13 Gly14 Gly15 Gly16 Ser17 Tyr18 Thr19 Tyr20 Tyr21 Pro22 Asp23 Ser24 Val25 Lys26 Gly27. The VHCDR2 sequence has a total of [number missing] complementary bits across all complementary positions. Thr13、Gly14、Gly15、Gly16、Ser17 and Tyr18Therefore, Thr11, Ile12, Thr19, Tyr20, Tyr21, Pro22, Asp23, Ser24, Val25, Lys26, and Gly27 can individually substitute any amino acid residue. For example, Thr11 can independently substitute Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val; Ile12 can independently substitute Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Thr19 can independently substitute Ala, Arg, Asn, Asx, Cys, Glu, Gln, Glx, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and Thr19 can independently substitute Ala, Arg, Asx ... n, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val; Tyr20 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val; Tyr21 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx. Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val; Pro22 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val; Asp23 can independently replace Ala, Arg, Asn, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pr o, Ser, Thr, Trp, Tyr, or Val; Ser24 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val; Val25 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;Lys26 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr; and Gly27 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.

[0415] VHCDR3 sequence A S P YLN YF DY (SEQ ID NO: 20) could be Ala Ser Pro Tyr Leu Asn Tyr Phe Asp Tyr And the number is Ala30 Ser31 Pro32 Tyr33 Leu34 Asn35 Tyr36 Phe37 Asp38 Tyr39 The VHCDR3 sequence has a total of [number] complementary bits across all complementary bits. Ser31、Tyr33、Leu34、Asn35、Asp38、Tyr39 Therefore, Ala30, Pro32, Tyr36, and Phe37 can individually substitute any amino acid residue. For example, Ala30 can independently substitute Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Pro32 can independently substitute Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val. l; Tyr36 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val; and Phe37 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val.

[0416] VHCDR3 sequence P YLN YF DY (SEQ ID NO: 11) can be Pro Tyr Leu Asn Tyr Phe Asp TyrAnd the model number is Pro32 Tyr33 Leu34 Asn35 Tyr36 Phe37 Asp38 Tyr39 The VHCDR3 sequence has a total of [number] complementary bits across all complementary bits. Tyr33、Leu34、Asn35、Asp38、Tyr39 Therefore, Pro32, Tyr36, and Phe37 can individually substitute any amino acid residue. For example, Tyr36 can independently substitute Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val; and Phe37 can independently substitute Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val.

[0417] VHCDR3 sequence S P YLN YF DY (SEQ ID NO: 23) can be Ser Pro Tyr Leu Asn Tyr Phe Asp Tyr And the number is Ser31 Pro32 Tyr33 Leu34 Asn35 Tyr36 Phe37 Asp38 Tyr39 The VHCDR3 sequence has a total of [number] complementary bits across all complementary bits. Ser31、Tyr33、Leu34、Asn35、Asp38、Tyr39 Therefore, Pro32, Tyr36, and Phe37 can individually substitute for any amino acid residue. For example, Pro32 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val; Tyr36 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val; and Phe37 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val.

[0418] In one embodiment, the P1801 variable region light chain sequence that defines the CDR showing the complementary bit is defined as follows:

[0419] EIVLTQSPATLSLSPGERATLSCRASQSISNNLHWYQQKPGQAPRLLIYYASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSNSWPLTFGQGTKLEIK (SEQ ID No: 6).

[0420] The VLCDR1 sequence RASQSISN was demonstrated. N LH (SEQ ID NO: 12) can also be written as Arg Ala Ser GlnSer Ile Ser Asn Asn Leu His, numbered Arg1 Ala2 Ser3 Gln4 Ser5 Ile6 Ser7Asn8 Asn9 Leu10 His11. The VLCDR1 sequence has a total of [number] complementary bits across all complementary bits. Asn9Therefore, Arg1, Ala2, Ser3, Gln4, Ser5, Ile6, Ser7, Asn8, Leu10, and His11 can individually substitute for any amino acid residue.For example, Arg1 can independently replace Ala, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Ala2 can independently replace Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Ser3 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Gl... x, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val; ln4 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Ser5 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val; Ile6 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Ser7 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val; Asn8 can independently replace Ala, Arg, Asp, Asx, Cys, Glu, Gln, Glx, Gly, and His. Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and Leu10 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and His11 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.

[0421] VLCDR2 sequence Y ASQSIS (SEQ ID NO: 13) can be Tyr Ala Ser Gln Ser Ile Ser And the number is Tyr12 Ala13 Ser14 Gln15 Ser16 Ile17 Ser18 The VLCDR2 sequence has a total of [number] complementary bits across all complementary bits. Tyr12 and Ser18 Therefore, Ala13, Ser14, Gln15, Ser16, and Ile17 can individually substitute for any amino acid residue. For example, Ala13 can independently substitute for Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Ser14 can independently substitute for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val; and Gln15 can independently substitute for Ala, Arg, Asn, Asp, Asx, Cys, Glu, Glx, Gl... y, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Ser16 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val; and Ile17 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.

[0422] VLCDR3 Serial QQ SN S W PLT (SEQ ID NO: 14) can be Gln Gln Sir Asn Ser Trp Pro LeuThr and numbered Gln20 Gln21 Ser22 Asn23 Ser24 Trp25 Pro26 Leu27 Thr28.

[0423] The VLCDR3 sequence has a total of complementary bits across all complementary bits. Ser22、Asn23、Trp25Therefore, Gln20, Gln21, Ser24, Pro26, Leu27, and Thr28 can individually substitute for any amino acid residue. For example, Gln20 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Gln21 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Ser24 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val. l; Pro26 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val; Leu27 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and Thr28 can independently replace Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val.

[0424] Table 15

[0425]

[0426] In the complementary sequences listed in Table 15, the amino acid residues presented in bold and underlined are constant across various proteins. The remaining amino acid residues in the sequence can individually substitute for any amino acid residue, such as Ala, Arg, Asn, Asp, Asx, Cys, Glu, Gln, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. The condition is that the bolded and underlined residues in the CDR are retained, allowing for the individual substitution of the remaining amino acid residues while maintaining function. Therefore, this disclosure contemplates numerous variations of the CDR sequence, provided that the bolded and underlined residues in the CDR are retained, and one or more of the remaining amino acid residues are substituted. In view of this functional definition, the inventors of this application have considered many different CDR sequences characterized by the bolded and underlined residues in the CDR, one or more substituted amino acids, and the retention of sequence function.

[0427] In consideration, any embodiment described herein can be implemented using any method, kit, reagent, or composition of this disclosure, and vice versa. Furthermore, the compositions of this disclosure can be used to implement the methods of this disclosure.

[0428] It should be understood that the specific embodiments described herein are illustrated by way of illustration and are not intended to limit the scope of this disclosure. The main features of this disclosure may be employed in various embodiments without departing from its scope. Those skilled in the art will recognize or be able to determine many equivalents of the specific procedures described herein using only conventional experimentation. These equivalents are all considered to be within the scope of this disclosure and are covered by the following claims.

[0429] All publications and patent applications mentioned in this specification indicate the level of skill of a person skilled in the art to which this disclosure pertains. All publications and patent applications are incorporated herein by reference to the extent that each individual publication or patent application is specifically and individually indicated to be incorporated by reference.

[0430] When used in conjunction with the term "comprising" in the claims and / or this specification, the word "a / an" may mean "one," but it also encompasses the meanings of "one or more," "at least one," and "one or more." Although this disclosure supports the definitions of "and / or" referring only to substitutes, the term "or" in the claims is used to mean "and / or" unless explicitly indicated as referring only to substitutes or that the substitutes are mutually exclusive. Throughout this application, the term "about" is used to indicate that a value includes the inherent bias of the apparatus, the method used to determine the value, or a bias that exists between individuals studied.

[0431] As used in this specification and claims, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional unlisted elements or method steps.

[0432] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and if the order is significant in a particular case, it also includes BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing this example, combinations containing one or more repeated items or terms are explicitly included, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those skilled in the art will understand that, unless otherwise apparent from the context, there is generally no limitation on the number of items or terms in any combination.

[0433] All compositions and / or methods disclosed and claimed herein can be prepared and performed according to this disclosure without excessive experimentation. Although the compositions and methods of this disclosure have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that variations may be made to the compositions and / or methods described herein, as well as the steps or order of steps of the methods, without departing from the concept, spirit, and scope of this disclosure. All such similar substitutions and modifications that will be apparent to those skilled in the art should be considered to be within the spirit, scope, and concept of this disclosure as defined in the appended claims.

Claims

1. An isolated antibody or its antigen-binding moiety that preferentially binds to PD-1, comprising: Includes the heavy chain complementarity determining region one (VHCDR1) of the amino acid sequences listed in SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 21; includes the heavy chain complementarity determining region two (VHCDR2) of the amino acid sequences listed in SEQ ID NO: 10; and / or includes the heavy chain complementarity determining region three (VHCDR3) of the amino acid sequences listed in SEQ ID NO: 11, SEQ ID NO: 20, or SEQ ID NO: 23; and Includes light chain complementarity determining region one (VLCDR1) of the amino acid sequence listed in SEQ ID NO: 12, light chain complementarity determining region two (VLCDR2) of the amino acid sequence listed in SEQ ID NO: 13, and / or light chain complementarity determining region three (VLCDR3) of the amino acid sequence listed in SEQ ID NO:

14.

2. A separated antibody or its antigen-binding moiety that preferentially binds to PD-1, comprising one or more paratopes, said one or more paratopes comprising: At least one of the following residues: Phe2, Ser5, Ser6 and Tyr7 of the heavy chain variable sequence (VHCDR1) of SEQ ID NO: 19; At least one of the following residues: Phe2, Ser5 and Tyr6 of the heavy chain variable sequence (VHCDR1) of SEQ ID NO: 21; At least one of the following residues: Thr13, Gly14, Gly15, Gly16, Ser17 and Tyr18 of the heavy chain variable sequence (VHCDR2) of SEQ ID NO: 10; At least one of the following residues: Tyr33, Leu34, Asn35, Asp38, and Tyr39 of the heavy chain variable sequence (VHCDR3) of SEQ ID NO: 11; or At least one of the following residues: Ser31, Tyr33, Leu34, Asn35, Asp38 and Tyr39 of the heavy chain variable sequence (VHCDR3) of SEQ ID NO:

20.

3. A separable antibody or antigen-binding moiety thereof that preferentially binds to PD-1, comprising one or more complementary sites, said one or more complementary sites comprising: At least one of the following residues: Asn9 of the light chain variable sequence (VLCDR1) of SEQ ID NO: 12; At least one of the following residues: Tyr12 and Ser18 of the heavy chain variable sequence (VLCDR2) of SEQ ID NO: 13; or At least one of the following residues: Ser22, Asn23 and Trp25 of the heavy chain variable sequence (VLCDR3) of SEQ ID NO:

14.

4. A separated antibody or antigen-binding moiety thereof that preferentially binds to PD-1, comprising one or more complementary sites, said one or more complementary sites comprising: At least one of the following residues: Phe2, Ser5, Ser6 and Tyr7 of the heavy chain variable sequence (VHCDR1) of SEQ ID NO: 19; At least one of the following residues: Phe2, Ser5 and Tyr6 of the heavy chain variable sequence (VHCDR1) of SEQ ID NO: 21; At least one of the following residues: Thr13, Gly14, Gly15, Gly16, Ser17 and Tyr18 of the heavy chain variable sequence (VHCDR2) of SEQ ID NO: 10; At least one of the following residues: Tyr33, Leu34, Asn35, Asp38 and Tyr39 of the heavy chain variable sequence (VHCDR3) of SEQ ID NO: 11; At least one of the following residues: Ser31, Tyr33, Leu34, Asn35, Asp38 and Tyr39 of the heavy chain variable sequence (VHCDR3) of SEQ ID NO: 20; At least one of the following residues: Asn9 of the light chain variable sequence (VLCDR1) of SEQ ID NO: 12; At least one of the following residues: Tyr12 and Ser18 of the heavy chain variable sequence (VLCDR2) of SEQ ID NO: 13; or At least one of the following residues: Ser22, Asn23 and Trp25 of the heavy chain variable sequence (VLCDR3) of SEQ ID NO:

14.

5. An isolated antibody or its antigen-binding moiety that preferentially binds to PD-1, comprising: (i) Contains the heavy chain variable region (VH) of SEQ ID NO: 9 and Contains the light chain variable region (VL) of SEQ ID NO: 6; or (ii) Containing the heavy chain variable region (VH) of SEQ ID NO: 18 and Contains the light chain variable region (VL) of SEQ ID NO: 6; or (iii) The heavy chain variable region (VH) containing SEQ ID NO: 19 and Contains the light chain variable region (VL) of SEQ ID NO: 6; or (iv) The heavy chain variable region (VH) containing SEQ ID NO: 21 and It contains the light chain variable region (VL) of SEQ ID NO:

6.

6. An isolated antibody or its antigen-binding moiety that preferentially binds to PD-1, comprising... (i) Containing the heavy chain variable region (VH) of SEQ ID NO: 5 or Contains the heavy chain variable region (VH) of SEQ ID NO: 22; and (ii) Contains the light chain variable region (VL) of SEQ ID NO:

6.

7. An isolated antibody or its antigen-binding moiety that preferentially binds to PD-1, comprising: Heavy chain complementarity-determining regions 1 (VHCDR1), 2 (VHCDR2), and / or 3 (VHCDR3) derived from the heavy chain variable region amino acid sequence listed in SEQ ID NO: 5; and Light chain complementarity-determining region 1 (VLCDR1), light chain complementarity-determining region 2 (VLCDR2) and / or light chain complementarity-determining region 3 (VLCDR3) obtained from the amino acid sequence of the light chain variable region listed in SEQ ID NO:

6.

8. The antibody or antigen-binding portion thereof according to any one of claims 1 to 7, further comprising a heavy chain constant region (HCCR) comprising the amino acid sequence listed in SEQ ID NO: 7; The HCCR may optionally include one, two, or three conserved amino acid substitutions.

9. The antibody or antigen-binding portion thereof according to any one of claims 1 to 7, further comprising a light chain constant region (LCCR) comprising the amino acid sequence of SEQ ID NO: 8; wherein the LCCR optionally comprises one, two or three conserved modified amino acid substitutions.

10. The antibody or antigen-binding portion thereof according to any one of claims 1 to 7, further comprising: a heavy chain constant region (HCCR) including the amino acid sequence listed in SEQ ID NO: 7, wherein the HCCR optionally includes one, two, or three conserved modified amino acid substitutions; and The light chain constant region (LCCR) includes the amino acid sequence of SEQ ID NO: 8, wherein the LCCR optionally includes one, two or three conserved amino acid substitutions.

11. The antibody or its antigen-binding portion according to claim 10, further comprising... The heavy chain signal peptide (HCSP) at the N-terminus of the VH comprises the amino acids listed in SEQ ID NO:

15.

12. The antibody or antigen-binding portion thereof according to claim 10, further comprising a light chain signal peptide (LCSP) at the N-terminus of the VL, the light chain signal peptide comprising the amino acids listed in SEQ ID NO:

16.

13. The antibody or its antigen-binding portion according to any one of claims 1 to 7, wherein the antibody is a human or humanized antibody.

14. The antibody or its antigen-binding portion according to any one of claims 1 to 7, wherein the antibody is an IgG4 isotype.

15. A pharmaceutical composition for treating, inhibiting, and / or suppressing solid tumors, comprising: (i) the antibody or its antigen-binding portion according to any one of claims 1 to 7; and (ii) Pharmaceutically acceptable carriers.

16. A polynucleotide molecule encoding an antibody or antigen-binding moiety according to any one of claims 1 to 7.

17. An expression vector comprising the polynucleotide molecule of claim 16, wherein the expression vector encoding LCSP does not contain introns.

18. A host cell comprising the expression vector according to claim 17.

19. A method for preventing, treating, and / or suppressing tumors in an individual, comprising the following steps: An effective amount of the antibody or antigen-binding moiety according to claims 1 to 7 is administered to the individual.

20. The method of claim 19, wherein the tumor comprises hepatocellular carcinoma, or a solid tumor comprising melanoma and / or renal cell carcinoma (RCC).

21. A method for regulating the activity of one or more T cells in an individual in vitro, ex vivo, and / or in vivo, the method comprising the following steps: The T cells are contacted with an antibody or its antigen-binding moiety that preferentially binds to human programmed death receptor 1 (hPD-1), wherein the antibody blocks and / or modulates the binding of human PD-L1 and / or human PD-L2 to hPD-1, and wherein the antibody or its antigen-binding moiety comprises: Includes the heavy chain complementarity-determining region 1 (VHCDR1) of the amino acid sequences listed in SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 21, includes the heavy chain complementarity-determining region 2 (VHCDR2) of the amino acid sequences listed in SEQ ID NO: 10, and / or includes the heavy chain complementarity-determining region 3 (VHCDR3) of the amino acid sequences listed in SEQ ID NO: 11, SEQ ID NO: 20, or SEQ ID NO: 23; and It includes light chain complementarity-determining region 1 (VLCDR1) of the amino acid sequence listed in SEQ ID NO: 12, light chain complementarity-determining region 2 (VLCDR2) of the amino acid sequence listed in SEQ ID NO: 13, and / or light chain complementarity-determining region 3 (VLCDR3) of the amino acid sequence listed in SEQ ID NO:

14.

22. The method of claim 21, wherein the antibody or its antigen-binding portion further comprises a heavy chain constant region (HCCR), the heavy chain constant region comprising the amino acid sequence listed in SEQ ID NO: 7, and the HCCR optionally comprising one, two or three conserved modified amino acid substitutions.

23. The method of claim 21, wherein the antibody or its antigen-binding portion further comprises a light chain constant region (LCCR), the light chain constant region comprising the amino acid sequence of SEQ ID NO: 8, and wherein the LCCR optionally comprises one, two, or three conserved amino acid substitutions.

24. The method of claim 21, wherein the one or more T cells comprise one or more natural killer cells, one or more cytotoxic T cells, one or more helper T cells, one or more monocytes, one or more memory T cells and / or one or more regulatory T cells.

25. The method of claim 21, wherein the antibody or antigen binding portion is at an IC50 concentration of about 1 nM or lower. 50 Blocking the binding of human PD-L1 and human PD-L2 to human PD-1.

26. A method for generating an antibody or antigen-binding partial vector construct, comprising: The host cells according to claim 18 are cultured under conditions that allow expression of the antibody or antigen-binding moiety construct; and The resulting antibody or antigen-binding vector construct is purified and / or recovered from the culture.

27. An antibody or its antigen-binding portion for treating solid tumors, comprising: a. Containing the heavy chain variable region (VH) of SEQ ID NO: 5 or the heavy chain variable region (VH) of SEQ ID NO: 22; b. Contains the light chain variable region (VL) of SEQ ID NO: 6; and Optionally, another therapeutic agent may be included.

28. The antibody or antigen-binding portion thereof according to claim 27, further comprising: a heavy chain constant region (HCCR) comprising the amino acid sequence listed in SEQ ID NO:7, wherein the HCCR optionally comprises one, two, or three conserved amino acid substitutions; and a light chain constant region (LCCR) comprising the amino acid sequence of SEQ ID NO:8, wherein the LCCR optionally comprises one, two, or three conserved amino acid substitutions; and Optionally, another therapeutic agent may be included.

29. The antibody or its antigen-binding portion according to claim 27, wherein the other therapeutic agent comprises P1101, P1801 and / or another anti-human PD1 antibody.

30. The antibody or its antigen-binding portion according to claim 28, wherein the other therapeutic agent comprises P1101, P1801 and / or another anti-human PD1 monoclonal antibody.

31. The antibody or its antigen-binding portion according to any one of claims 28 to 30, wherein the solid tumor comprises hepatocellular carcinoma, renal cell carcinoma, and / or melanoma.

32. A combination for treating, inhibiting, preventing, and / or suppressing a solid tumor in an individual, wherein the combination comprises... Antibody or its antigen-binding portion; and Another treatment, The antibody or its antigen-binding portion comprises: a. Containing the heavy chain variable region (VH) of SEQ ID NO: 5 or the heavy chain variable region (VH) of SEQ ID NO: 22; and b. Contains the light chain variable region (VL) of SEQ ID NO:

6.

33. The combination of claim 32, wherein the antibody or its antigen-binding portion further comprises: a heavy chain constant region (HCCR) comprising the amino acid sequence listed in SEQ ID NO: 7, wherein the HCCR optionally comprises one, two or three conserved amino acid substitutions; and a light chain constant region (LCCR) comprising the amino acid sequence of SEQ ID NO: 8, wherein the LCCR optionally comprises one, two or three conserved amino acid substitutions.

34. The combination according to any one of claims 32 to 33, wherein the therapeutic agent comprises P1101, P1801 and / or another anti-human PD1 monoclonal antibody.

35. The combination of claim 34, wherein the P1101, P1801 and / or another anti-human PD1 monoclonal antibody are administered sequentially and / or approximately simultaneously.

36. The combination of claim 35, wherein P1101 and / or P1801 is administered prior to another anti-human PD1 monoclonal antibody.

37. The combination according to claim 32 or 33, wherein the antibody or its antigen-binding portion and the other therapeutic agent are administered as a single composition or as separate compositions.

38. The combination of any one of claims 32 to 37, wherein the solid tumor comprises hepatocellular carcinoma, melanoma, and / or renal cell carcinoma.

39. A recombinant protein comprising... (i) the antibody or antigen-binding moiety according to claim 1; and (ii) An optional tag sequence for expressing detection and / or purification.

40. The isolated antibody or its antigen-binding portion according to any one of claims 1 to 2, 5 to 7 or 27, in a method of treating cancer, the method comprising administering the isolated antibody or its antigen-binding portion to an individual.

41. The isolated antibody or its antigen-binding portion according to claim 40, wherein the cancer comprises hepatocellular carcinoma, melanoma, and / or renal cell carcinoma.

42. Use of an isolated antibody or its antigen-binding portion according to any one of claims 1 to 2, 5 to 7 or 27 for the manufacture of a medicament for therapeutic and / or preventive treatment of cancer.

43. Use of the isolated antibody or its antigen-binding portion according to claim 42, wherein the cancer comprises hepatocellular carcinoma, melanoma, and / or renal cell carcinoma.

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