Immunoconjugate of Anti-PD-1 antibody and cytokine il-12 mutant protein, and use thereof

Abstract

The present invention relates to an immunoconjugate, and in particular, to an immunoconjugate containing an antigen-binding region binding to PD-1, and a mutant IL-12 protein. Also provided are a polynucleotide molecule encoding the immunoconjugate, and a vector and host cell containing such polynucleotide molecules. Further provided are a method for generating the immunoconjugate, a pharmaceutical composition containing same, a treatment method by using same, and the use thereof.

Description

Immunoconjugates of anti-pd-1 antibodies and cytokine il-12 muteins and uses thereof

[0001] Cross-reference to Related Applications

[0002] This application is based on and claims priority to Chinese Patent Application No. 202410741283.9, filed on June 7, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to immunoconjugates, in particular immunoconjugates comprising an antigen binding region that binds PD-1 and a mutant IL-12 protein. In addition, the present application relates to polynucleotide molecules encoding the immunoconjugates and vectors and host cells comprising such polynucleotide molecules. The present application further relates to methods for producing the immunoconjugates, pharmaceutical compositions comprising them, and therapeutic methods and uses applying the same. BACKGROUND

[0004] Programmed cell death protein 1 (PD-1 or CD279) is an inhibitory member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is a cell surface receptor and is expressed on activated B cells, T cells, and myeloid cells. PD-1 is a monomeric type I transmembrane protein composed of an immunoglobulin variable-like extracellular domain and a cytoplasmic domain containing an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). Two ligands of PD-1, PD-L1 and PD-L2, have been identified, which downregulate T cell activation upon binding to PD-1. Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1 but not to other CD28 family members. One ligand of PD-1, PD-L1, is highly expressed in a variety of human cancers, and thus the interaction between PD-1 and PD-L1 leads to reduced tumor-infiltrating lymphocytes, decreased T cell receptor-mediated proliferation, and immune evasion of cancerous cells. The advent of PD-1 monoclonal antibodies has brought new hope to tumor immunotherapy and has revolutionized the concept of cancer treatment. However, only about 20% of patients with solid tumors benefit from treatment with PD-1 antibodies. This is because the monoclonal antibodies against PD-1 mainly work by relieving the PD-1-mediated T cell immune suppression. In theory, this drug action mechanism is relatively passive, and if the killing activity of the patient's killer T cells is relatively low, simply relieving immune suppression is not enough to achieve the purpose of eliminating tumor cells. Therefore, there is a high unmet need for the development of new immunotherapeutic drugs for a large number of solid tumor patients who do not respond or are resistant to PD-1 monoclonal antibody treatment.

[0005] Interleukin 12 (IL-12) is a cytokine with multiple functions in the immune system. IL-12 is a heterodimer containing two subunits, p35 (encoded by the IL-12A gene) and p40 (encoded by the IL-12B gene). IL-12 binds to the cross-linked heterodimeric IL-12 receptor (IL-12R) chains, IL-12Rβ1 and IL-12Rβ2. IL-12R is upregulated by T cell receptor (TCR) activation, thereby enhancing the sensitivity of T cells to IL-12 stimulation. After IL-12 binds to IL-12R, STAT4 is phosphorylated (pSTAT4), which in turn promotes CD8 + T cells, CD4 + T cells and NK cells produce and secrete interferon gamma (IFNg) and enhance the killing ability of the above-mentioned cells against tumor cells.

[0006] Preclinical models show that IL-12 promotes anti-tumor immunity by directly acting on T cells and NK cells, and indirectly acting on antigen-presenting cells in the tumor microenvironment (TME). However, preclinical and clinical studies also show that IL-12R agonists can have strong dose-limiting toxicities. These studies speculate that significant toxicity can be due to over-activation of lymphocytes (especially T cells and NK cells) by IL-12, stimulating the release of inflammatory factors.

[0007] Previous studies targeted cytokines including IL-15 and IFN to PD-1 positive cells by fusing anti-PD-1 antibodies with cytokine proteins. However, this targeting has limited effect on improving systemic toxicity of cytokines, and it cannot fundamentally solve the problem of insufficient clinical efficacy due to dose-limiting toxicity. For such a highly active cytokine as IL-12, using its mutant (partial agonist) to construct an immunoconjugate can more effectively solve the problem of high toxicity and low killing of IL-12, for example, constructing an immunoconjugate of IL-12 and PD-1 antibody.

[0008] However, there is still a need for improved IL-12 variants to address the toxicity and killing efficiency of cytokines, as well as related immunoconjugates, such as immunoconjugates of IL-12 and PD-1 antibodies. SUMMARY

[0009] The present application develops an IL-12 variant, and an immunoconjugate molecule targeting PD-1 and IL-12 receptor based thereon.

[0010] The immunoconjugate of the present application has the following advantages:

[0011] (1) retain the immune checkpoint function of the PD-1 antibody, so that the patient's immune system is not inhibited;

[0012] (2) block the binding of PD-L1 to PD-1 in cells;

[0013] (3) does not cause downstream signal activation of peripheral T cells in the peripheral environment in vivo, increasing the safety of the periphery;

[0014] (4) does not cause proliferation of peripheral T cells in the systemic circulation in vivo, reducing systemic toxicity;

[0015] (5) has obvious selectivity for T cells with different PD-1 expression amounts, so there is a larger safety window in vivo;

[0016] (6) has a certain proliferative effect on CD4 + T and CD8 + T cells, but the proliferative effect is significantly reduced compared to that of the wild-type IL-12-Fc molecule, thereby reducing toxicity in vivo;

[0017] (7) can inhibit tumor growth and has in vivo safety, such as no effect on body weight. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 shows the molecular structure of the immunoconjugate of the present application.

[0019] Figure 2 shows that the PD-1 antibody end of the αPD-1 / IL-12m immunoconjugate (1152, 1167) of the present study has a biological activity (i.e., PD-1 / PD-L1 signal pathway blocking effect) comparable to that of the parent anti-PD-1 monoclonal antibody IBI308 (Sintilimab).

[0020] Figure 3 shows that the αPD-1 / IL-12m immunoconjugate of the present study does not stimulate the downstream pSTAT4 signal of activated T lymphocytes in unpreactivated PBMC.

[0021] Figure 4 shows that the αPD-1 / IL-12m immunoconjugate of the present study has no obvious proliferative effect on CD4 + T and CD8 + T cells in unpreactivated PBMC.

[0022] Figure 5 shows that the αPD-1 / IL-12m immunoconjugate of the present study has a proliferative effect on T cells (PD1 high CD4 + T and PD1 high CD8​​+ T) and low PD-1 expressing T cells (PD1 low CD4 + T and PD1 low CD8 + molecular activity in T cells (T) and low PD-1 expressing T cells (PD1

[0023] Figure 6 shows the proliferation of CD4 + T and CD8 + T cells by the aPD-1 / IL-12m immunoconjugate of the present study.

[0024] Figure 7 shows the molecular activity in high PD-1 expressing T cells (PD1 high CD4 + T and PD1 high CD8 + T) and low PD-1 expressing T cells (PD1 low CD4 + T and PD1 low CD8 + molecular activity in T cells (T) and low PD-1 expressing T cells (PD1

[0025] Figure 8 shows the molecular activity in high PD-1 expressing T cells (PD1 high CD4 + T and PD1 high CD8 + T) and low PD-1 expressing T cells (PD1 low CD4 + T and PD1 low CD8 + molecular activity in T cells (T) and low PD-1 expressing T cells (PD1

[0026] Figure 9 shows the inhibition of tumor growth (Figure 9A) and the effect on the weight of the mice (Figure 9B) by the mouse surrogate molecules of the aPD-1 / IL-12m immunoconjugate of the present study.

[0027] Figure 10 shows the inhibition of tumor growth (Figure 10A) and the effect on the weight of the mice (Figure 10B) by the mouse surrogate molecules of the aPD-1 / IL-12m immunoconjugate of the present study.

[0028] Figure 11 shows the inhibition of tumor growth (Figure 11A) and the effect on the weight of the mice (Figure 11B) by the mouse surrogate molecules of the aPD-1 / IL-12m immunoconjugate of the present study.

[0029] Figure 12 shows the inhibition of tumor growth (Figure 12A) and the effect on mouse weight (Figure 12B) by the aPD-l / IL-12m immunoconjugate of the present study.

[0030] Figure 13 shows the inhibition of tumor growth (Figure 13A) and the effect on mouse weight (Figure 13B) by the aPD-l / IL-12m immunoconjugate of the present study.

[0031] Figure 14 shows the inhibition of tumor growth (Figure 14A) and the effect on mouse weight (Figure 14B) by the aPD-l / IL-12m immunoconjugate of the present study.

[0032] Definitions

[0033] Before the present application is described in detail, it is to be understood that this application is not limited to the particular methodology, protocols, and reagents described herein as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present application which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0034] To interpret the description, the following definitions will apply and whenever appropriate, terms used in the singular will include the plural and vice versa. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0035] The term "about" used in connection with a numerical value means a range of numerical values that has a lower limit that is 5% (e.g., 4%, 3%, 2%, or 1%) less than the specified numerical value and an upper limit that is 5% (e.g., 4%, 3%, 2%, or 1%) greater than the specified numerical value.

[0036] As used herein, the term "and / or" means either or both of the items it connects.

[0037] As used herein, the term "comprising" or "including," means including, but not limited to, whatever follows the term. In the present description, when using the term "comprising" or "including," the items or steps are not meant to be limited to the listed items or steps, unless otherwise specified. For example, when referring to an IL-12 mutein "comprising" or "including" a certain mutation or combination of mutations, it is also intended to encompass an IL-12 mutein having only the mutation or combination of mutations.

[0038] In the present context, wild-type "interleukin-12" or "IL-12" refers to the parent IL-12 protein, preferably a naturally occurring IL-12 protein, such as one derived from human, mouse, rat, non-human primate, including both unprocessed (e.g. without signal peptide removal) and processed (e.g. with signal peptide removal) forms, which serves as the template for introducing mutations or combinations of mutations of the present application.

[0039] In the present context, an amino acid mutation can be an amino acid substitution, deletion, insertion and / or addition. Any combination of substitutions, deletions, insertions and additions can be made to obtain the final mutant protein with desired properties. Amino acid deletions and insertions include deletions and insertions at the amino and / or carboxyl terminus of a polypeptide sequence, as well as within the polypeptide sequence. For example, deletions can be made at positions 15-18 of the p40 subunit of full-length human IL-12. In some embodiments, the amino acid mutation is an amino acid substitution, such as a combination of single amino acid substitutions or a replacement of a segment of the amino acid sequence. For example, positions 259-265 (corresponding to SKREKKD) of the p40 subunit of wild-type IL-12 can be substituted in whole or in part with a different sequence (e.g. DNTEG). As another example, one or several mutations can be introduced by substitution to weaken the binding of IL-12 to its receptor IL-12Rβ1.

[0040] In the present application, when referring to an amino acid position in the p40 subunit of an IL-12 protein (IL-12p40) or a sequence segment thereof, it is determined by reference to the amino acid sequence of the p40 subunit of wild-type human IL-12 protein (also referred to as IL-12p40 WT ) of SEQ ID NO: 4. The corresponding amino acid position on other IL-12p40 proteins or polypeptides (including full-length sequences or truncated fragments) can be identified by amino acid sequence alignment with SEQ ID NO: 4. Thus, in the present application, unless otherwise specified, the amino acid position of an IL-12p40 protein or polypeptide is determined according to the amino acid position numbering of SEQ ID NO: 4. For example, when referring to "K84", it refers to the lysine residue K at position 84 of SEQ ID NO: 4, or the amino acid residue at the corresponding position on other IL-12p40 polypeptide sequences upon alignment. Meanwhile, for ease of understanding and comparison, when the mutations of the present application involve site truncation or deletion of certain specific segments (e.g. the 4 amino acid residues at positions 15-18 of SEQ ID NO: 4), the amino acid residue numbering outside the given mutation region and its mutation manner remains unchanged, e.g. after deletion of the 4 amino acid residues at positions 15-18 of SEQ ID NO: 4, the positions 15-18 in the numbering are no longer assigned, while the next amino acid residue position number following it is still 19.

[0041] In the present application, when referring to an amino acid position in the p35 subunit of an IL-12 protein (IL-12p35) or a sequence segment thereof, it is determined by reference to the amino acid sequence of the p35 subunit of the wild-type human IL-12 protein (also referred to as IL-12p35 WT ) of SEQ ID NO: 3. The corresponding amino acid position on other IL-12p35 proteins or polypeptides (including full-length sequences or truncated fragments) can be identified by amino acid sequence alignment with SEQ ID NO: 3. Thus, in the present application, unless otherwise specified, the amino acid position of an IL-12p35 protein or polypeptide is determined according to the amino acid position numbering of SEQ ID NO: 3. For example, when referring to “K170”, it refers to the lysine residue K at position 170 of SEQ ID NO: 3, or the amino acid residue at the corresponding position on other IL-12p35 polypeptide sequences as aligned.

[0042] The sequence alignment performed for the determination of amino acid positions can be performed using the Basic Local Alignment Search Tool available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi with default parameters.

[0043] In this document, when referring to an IL-12 mutein (e.g. IL-12p35 or IL-12p40 subunit mutein), a single amino acid substitution is described in the following manner: [original amino acid residue / position / substituted amino acid residue]. For example, a lysine substitution at position 170 to alanine can be denoted as K170A. When there can be multiple alternative amino acid substitutions at a given position (e.g. A or S at K170), the amino acid substitution can be denoted as: K35S / A. Correspondingly, individual single amino acid substitutions can be connected by the symbol “+” or “&” or “-” to indicate a combination of mutations at multiple given positions. For example, a combination of mutations at positions K170A and K84E can be denoted as: K84E+K170A, or K84E-K170A.

[0044] In this document, when referring to IL-12 muteins (e.g., IL-12p35 or IL-12p40 subunit muteins), amino acid sequence segment substitutions / replacements are described in the following manner: [(original amino acid sequence segment) / (position) / (amino acid sequence segment that replaces)]. For example, the substitution of the sequence segment "(SKREKKD)" at positions 259-265 of the IL-12p40 protein with "(DNTEG)" can be denoted as ((SKREKKD) / (259-265) / (DNTEG). Alternatively, the substitution, replacement is expressed as "the substitution (or mutation) of 259-265 (SKREKKD) to (DNTEG) of IL-12p40" or "the substitution (or mutation) of SKREKKD to DNTEG of IL-12p40".

[0045] It is noted that in applying the above description, the amino acid at the corresponding position in other IL-12p35 or IL-12p40 polypeptide sequences aligned thereto as a parent can be different from the amino acid at the corresponding position of SEQ ID NO: 3 or SEQ ID NO: 4, but the mutation indicated can still be made.

[0046] In this document, "percentage of sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window, wherein the framework sequences (e.g., SEQ ID NO: 3 or SEQ ID NO: 4) are compared. Optimal alignment of sequences for comparison can be conducted by using any method of alignment of sequences. Alignment methods suitable for determining percent sequence identity include those described by Altschul et al., Nuc. Acids Res. 25: 3389-402, 1977 and Altschul et al., J. Mol. Biol. 215: 403-10, 1990. Software for determining percent sequence identity can be accessed through the National Center for Biotechnology Information. For purposes of this application, the percentage of identity is determined using the Basic Local Alignment Search Tool, available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi, using default parameters.

[0047] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the biological function of a protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Typical conservative amino acid substitutions refer to the substitution of one amino acid for another amino acid having similar chemical properties (e.g., charge or hydrophobicity). Tables of functional, conservative amino acid substitutions that are well known in the art provide conservative substitutions of functionally similar amino acids. It is well within the skill in the art to provide a table of conservative substitutions of functionally similar amino acids. The following lists 8 groups containing amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M).

[0048] For example, a wild-type IL-12p40 subunit can have conservative amino acid substitutions, or only conservative amino acid substitutions, relative to SEQ ID NO: 4, and in a preferred embodiment, the conservative substitutions are no more than 10 amino acid residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 residues. For another example, a mutant IL-12p40 subunit of the application can have conservative amino acid substitutions, or only conservative amino acid substitutions, relative to an IL-12p40 mutant protein sequence specifically given herein (e.g., SEQ ID NO: 11, 24, or 25), and in a preferred embodiment, the conservative substitutions are no more than 10 amino acid residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 residues.

[0049] As used herein, the terms "against," "binds," or "binds specifically" mean that the binding is selective for the target or antigen and can be distinguished from unwanted or non-specific interactions. The ability of a binding site to bind to a particular target or antigen can be determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as by radioimmunoassay (RIA) or biological film- based thin layer interference assay or MSD assay or surface plasmon resonance (SPR) method.

[0050] "Affinity" or "binding affinity" can be used to reflect the intrinsic binding capacity of the interaction between the members of a binding pair. The affinity of a molecule X for its binding partner Y can be represented by the equilibrium dissociation constant (K D ), which is the dissociation rate constant and the association rate constant (k dis and k onThe ratio of the dissociation constant (KD) to the association rate constant (ka). The binding affinity can be measured by common methods known in the art. One particular method for measuring affinity is the SPR affinity assay technique or the BLI assay technique or the ForteBio Kinetic Binding Assay herein.

[0051] When referring herein to "first" "second", this is merely to distinguish between two domains or two chains, without indicating the position of the two domains in any way.

[0052] As used herein, "antigen binding region that binds to PD-1", "antigen binding region that specifically binds to PD-1" are used interchangeably to refer to an antigen binding region that confers on the molecule comprising it the ability to bind to PD-1 with appropriate affinity.

[0053] The term "antigen binding region" as used herein refers to any portion of an antibody or antigen binding fragment thereof, e.g. a multispecific antibody or bispecific antibody or immunoconjugate, that binds to a particular target or antigen. The antigen binding region can be, for example, the antibody or immunoglobulin itself or an antibody fragment. Such an antigen binding region can or can not have a tertiary structure independent of the remainder of the multispecific antibody or bispecific antibody or immunoconjugate, and can or can not bind its antigen / epitope as a separate entity.

[0054] Effector cells include effector T cells (T lymphocytes), e.g. CD4 + T cells, CD8 + T cells, Th1, Th2 and regulatory T cells (Tregs). Effector cells can also include natural killer cells, macrophages, granulocytes, plasma cells or B cells (lymphocytes).

[0055] General information on the nucleotide sequences of human immunoglobulin light and heavy chains is given in Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0056] The term "vector", as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector associated with a host cell into which it has been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".

[0057] The terms "whole antibody" or "full-length antibody" are used interchangeably herein to refer to an antibody molecule having a native immunoglobulin molecule structure. In the case of a conventional four-chain IgG antibody, the full-length antibody comprises two heavy chains (H) and two light chains (L) which are interconnected by disulfide bonds. In the case of a heavy chain antibody which has only heavy chains but lacks light chains, the full-length antibody comprises two heavy chains (H) which are interconnected by disulfide bonds. For a conventional four-chain IgG antibody, the full-length antibody heavy chain is typically composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, wherein the heavy chain constant region comprises at least three domains, CH1, CH2 and CH3. The full-length antibody light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region, wherein the light chain constant region comprises one domain, CL. Each heavy chain variable region VH and each light chain variable region VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0058] The term "antibody fragment" includes a portion of an intact antibody. In preferred embodiments, the antibody fragment is an antigen-binding fragment.

[0059] The term "antigen-binding fragment" of an antibody is a molecule other than a full-length antibody that comprises a portion of a full-length antibody that is capable of binding to an antigen bound by the full-length antibody or competing with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment is derived) for binding to the antigen. Antigen-binding fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody, single-domain antibody (sdAb), nanobody. For example, Fab fragments can be obtained by papain digestion of a full-length antibody. In addition, pepsin digestion of a full-length antibody under denaturing conditions generates F(ab')2, which is a dimer of two Fab' fragments linked by a disulfide bond from the hinge region of each of the Fab' fragments. F(ab')2 can be reduced under denaturing conditions to break the disulfide bond in the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab that has an additional hinge region. Fv fragments consist of the VL and VH domains of a single arm of an antibody. The two domains of the Fv fragment, VL and VH, can be coded for by separate genes, but they can be brought into a single protein chain by recombinant methods using a synthetic linker that enables them to be made as a single protein chain, and in which the VL and VH regions pair to form a monovalent molecule. This single chain Fv (scFv) is a monospecific antibody fragment.

[0060] "Fab fragment" or "Fab" are used interchangeably herein to refer to an immunoglobulin fragment that consists of two polypeptide chains, comprising an immunoglobulin heavy chain variable domain VH, a heavy chain constant domain CHI, a light chain variable domain VL, and a light chain constant domain CL, wherein one polypeptide chain comprises, from N- to C-terminus, VH and one constant region selected from CHI and CL, and the other polypeptide chain comprises, from N- to C-terminus, VL and the other constant region selected from CL and CHI, wherein the VH domain and the VL domain pair to form an antigen binding site. In the present context, the Fab polypeptide chain comprising the heavy chain constant domain CHI is also referred to as "Fab heavy chain"; correspondingly, the Fab polypeptide chain comprising the light chain constant domain CL is also referred to as "Fab light chain".

[0061] A "complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is hypervariable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contacts"). CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of a heavy chain and light chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. CDRs located within the variable domain of an antibody heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while CDRs located within the variable domain of an antibody light chain are referred to as LCDR1, LCDR2, and LCDR3. The precise amino acid sequence boundaries of each CDR in a given light chain variable region or heavy chain variable region amino acid sequence can be determined using any of a number of well-known antibody CDR assignment schemes, or combinations thereof, including, for example: Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al., The classes of amino acid sequences and their correlation with protein structure and evolution (1989) Nature 342:877-883; Al-Lazikani et al., Standard conformations for the canonical structures of immunoglobulins, Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (imgt.cines.fr / ), and North CDR definitions based on affinity propagation clustering with a large number of crystal structures. Unless otherwise specified, in the present application, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways.CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence, such as any of the exemplary CDRs of the application.

[0062] In some embodiments, the CDRs of the heavy chain variable region of an antibody of the application are determined according to AbM. In some embodiments, the CDRs of the light chain variable region of an antibody of the application are determined according to Kabat.

[0063] In one embodiment, the HCDR1 of the application is defined according to the AbM rules, the HCDR2, HCDR3 are defined according to the Kabat rules, and the LCDRs are defined according to the Kabat rules, respectively:

[0064] The term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native-sequence Fc regions and variant Fc regions. Native immunoglobulin "Fc domains" contain two or three constant domains, i.e., CH2, CH3, and optionally CH4 domains. For example, in native antibodies, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 and CH3 domains) of a heavy chain derived from an IgG, IgA, and IgD class antibody; or the second, third, and fourth constant domains (CH2, CH3, and CH4 domains) of a heavy chain derived from an IgM and IgE class antibody. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or heavy chain constant region is according to the EU numbering scheme (also referred to as the EU index) as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. However, the C-terminal lysine (Lys447) of the Fc region can or can not be present. Two Fc regions can dimerize to constitute a dimeric Fc, two different Fc heterodimerize to form a heterodimeric Fc. In this context, the terms "Fc region", "Fc portion", and "dimeric Fc (e.g., heterodimeric Fc)" do not include the heavy chain variable region VH and light chain variable region VL, and the heavy chain constant region CH1 and light chain constant region CL of an immunoglobulin, but can in some cases include the hinge region N-terminal to the heavy chain constant region. In one embodiment, a human IgG heavy chain Fc region extends from Asp221, or from Cys226, or from Asp231, to the carboxy-terminus of the heavy chain. In this context, when referring to an Fc region, unless specified otherwise, it refers to an Fc region extending from Asp221 to the carboxy-terminus of the heavy chain.

[0065] In one embodiment, the Fc region is derived from a human Fc region. In one embodiment, the Fc region comprises a portion of a human constant region. Antibody Fc regions are directly involved in complement activation, Clq binding, C3 activation, and Fc receptor binding. In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region is of the human IgG4 subclass. In one embodiment, the Fc region is of the human IgGl subclass. In one embodiment, the Fc region is a Fc region of human IgGl, IgG2, IgG3, or IgG4.

[0066] In the present context, "heterodimeric Fc scaffold" refers to a scaffold comprising or formed upon dimerization of two different Fc regions, which can be linked at their N- or C-terminus to a domain that binds an antigen (e.g. a heavy and / or light chain variable region of an antibody or an antigen binding fragment of an antibody that can bind to a target molecule, or a soluble portion of a ligand or receptor that can bind to a target molecule) for the construction of a multispecific antibody, e.g. a bispecific antibody.

[0067] The term "CH1 region" refers to the portion of an antibody heavy chain polypeptide that extends from EU position 118 to EU position 220 (EU numbering system).

[0068] In some embodiments, CH1 can comprise a portion of the hinge region. In some embodiments, the Fc region can comprise a portion of the hinge region. In the present context, specific amino acid residues of an antibody Fc region are described according to the EU numbering system.

[0069] The term "linker" as used herein refers to any molecule that enables the direct linkage of different moieties of an immunoconjugate. Examples of linkers that establish a covalent linkage between different moieties of an immunoconjugate include peptide linkers and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol, polypropylene glycol. The term "peptide linker" according to the present application refers to a sequence of amino acids, wherein said sequence links together the amino acid sequences of the individual moieties of an immunoconjugate. For example, a peptide linker can link an IL-12 mutein to an Fc region or fragment thereof. For example, a peptide linker can also link the two subunits of an IL-12 mutein. Also for example, a peptide linker can also link two Fab fragments that specifically bind to PD-1, e.g. the heavy chains of two Fab fragments. Preferably, the peptide linker has a length that is sufficient to link two entities in such a way that they maintain their conformation relative to each other in a way that the desired activity is not hampered. The peptide linker can or can not predominantly comprise the following amino acid residues: Gly, Ser, Ala, or Thr. Useful linkers include glycine linkers, e.g. (G) nwherein n is an integer of at least 1 (and preferably 2, 3, 4, 5, 6, 7, 8, 9, 10). Useful linkers include glycine-serine polymers, including, for example, (GS) n (GSGGS) n (SEQ ID NO: 48), (GGGGS) n (SEQ ID NO: 49), (GGGS) n (SEQ ID NO: 50), and (GGGGS) n G (SEQ ID NO: 51), wherein n is an integer of at least 1 (and preferably 2, 3, 4, 5, 6, 7, 8, 9, 10). Useful linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. A hinge region of an antibody, or a portion thereof (e.g., a partial hinge region or a variant thereof), can also serve as a linker. In some embodiments, the linker comprises a partial hinge region (or one amino acid of a hinge region) and a glycine-serine polymer.

[0070] The antigen binding region in the immunoconjugate of the application can be an antigen binding region of a humanized antibody, a human antibody, or a chimeric antibody.

[0071] As used herein, the terms "first" and "second" with respect to Fc regions or monomers, etc., are used to facilitate differentiation when there is more than one of each class of module. The use of these terms is not intended to impart a particular order or orientation of the portions in the immunoconjugate unless explicitly so stated.

[0072] As used herein, the term "immunoconjugate" refers to a molecule comprising at least one IL-12 mutein and at least one antigen binding region that specifically binds PD-1. "Linked" means that the various portions of the immunoconjugate (e.g., the antigen binding region, the IL-12 mutein, or subunit variants thereof) are linked, e.g., by a peptide bond, either directly or via one or more linkers (e.g., a peptide linker).

[0073] The term "therapeutic agent" as described herein encompasses any substance that is effective in the prevention or treatment of a tumor (e.g., a cancer), including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents (e.g., immunosuppressive agents).

[0074] The term "effective amount" refers to the amount or dose of an antibody or fragment or composition or combination product of the application, which, upon single or multiple dose administration to a patient, exerts the intended effect in the patient in need of treatment or prevention. An "effective amount" can encompass a "therapeutically effective amount" or a "prophylactically effective amount."

[0075] A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount is also one in which any toxic or detrimental effects of the immunoconjugate or composition or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 40%, even more preferably by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100% relative to an untreated subject.

[0076] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Generally, the prophylactic effective amount will be less than the therapeutically effective amount, since the prophylactic dose is used before symptoms of the disease are apparent and not during an established disease state.

[0077] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of the original cell which carries the nucleic acid content. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny can not be completely identical to the parent cell both in nucleic acid content and in physical characteristics due to, e.g., mutations. Mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell are included herein.

[0078] The term "label" as used herein refers to a compound or composition that is conjugated or fused directly or indirectly to a reagent, such as a polynucleotide probe or antibody, and that facilitates detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, can catalyze chemical changes to a detectable substrate compound or composition. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling by conjugating the probe or antibody to a secondary reagent which itself is labeled with a detectable substance.

[0079] An "individual" or "subject" includes a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0080] The term "anti-tumor effect" refers to a biological effect that can be exhibited by a variety of means, including, but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival. In some embodiments, an anti-tumor effect also relates to an anti-tumor effect without a reduction in the subject's body weight.

[0081] The terms "tumor" and "cancer" are used interchangeably herein to encompass solid tumors and hematological tumors (encompassing lymphomas).

[0082] The term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. In certain embodiments, cancers suitable for treatment by the antibodies of the application include solid tumors or hematological tumors (encompassing lymphomas), etc., including metastatic forms of cancer. The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive as referred to herein.

[0083] The term "pharmaceutically acceptable excipient" refers to a diluent, adjuvant (such as Freund's adjuvant (complete and incomplete)), excipient, carrier or stabilizer, etc., that is administered in conjunction with an active ingredient.

[0084] The term "pharmaceutical composition" refers to a composition that is in a form suitable for administration into a subject and that does not contain additional ingredients that are biologically damaging to the subject.

[0085] The term "pharmaceutical combination" or "combination product" refers to a non-fixed combination or a fixed combination, including but not limited to a kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) an immunoconjugate of the application, and (ii) another therapeutic agent) are administered to the patient as separate entities, either simultaneously, without specific time limitations, or sequentially with no specific time limitations, wherein such administration provides therapeutically effective levels of the two or more active agents in the patient. The term "fixed combination" means that the two or more active agents are administered to the patient as a single entity. Preferably, the dosage and / or time intervals for the two or more active agents are selected so that a synergistic effect is achieved. The individual components can each be present in the form of separate formulations that are administered simultaneously, sequentially or in overlapping time intervals.

[0086] The term "combination therapy" refers to the administration of two or more therapeutic agents, or treatment modalities, e.g., radiotherapy or surgery, to treat a disease described herein. Such administration

[0087] As used herein, "treatment" refers to slowing, interrupting, arresting, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

[0088] As used herein, "prevention" includes inhibition of the onset or progression of a disease or disorder, or symptoms of a particular disease or disorder. In some embodiments, a subject with a family history of cancer is a candidate for a prophylactic regimen. Typically, in the context of cancer, the term "prevention" refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0089] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector associated with a host cell into which it has been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0090] "Subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as from a fresh, frozen and / or preserved organ or tissue sample or biopsy or puncture sample; blood or any blood component; a body fluid, such as cerebrospinal fluid, amniotic fluid (amniotic water), peritoneal fluid (ascites), or interstitial fluid; a cell from a subject at any time during gestation or development. The tissue sample can contain compounds not naturally admixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. Specific Embodiments

[0091] I. IL-12 Mutant Proteins

[0092] In some embodiments, provided herein are interleukin 12 (IL-12) variants or mutants. IL-12 variants or mutants are also referred to herein as IL-12 mutant proteins.

[0093] IL-12 is a heterodimer comprising two subunits, p35 (also known as IL-12a; which is encoded by the IL-12A gene) and p40 (also known as IL-12b; which is encoded by the IL-12B gene). The two IL-12 subunits can form an inter-subunit disulfide bond between C177 of p40 and C74 of p35, or the two IL-12 subunits can be linked by a linker.

[0094] The amino acid sequence of the mature wild-type human IL-12 p35 subunit comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the IL-12 p35 subunit is produced from a full-length p35 polypeptide that also includes a 22 amino acid signal peptide that is cleaved during intracellular processing of the initially translated precursor protein. The full-length human p35 amino acid sequence including the signal peptide is available under UniProt accession number P29459, e.g., which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 38.

[0095] The amino acid sequence of the mature wild-type human IL-12 p40 subunit comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the IL-12 p40 subunit is produced from a full-length p40 polypeptide that also includes a 22 amino acid signal peptide that is cleaved during intracellular processing of the initially translated precursor protein. The full-length human p40 amino acid sequence including the signal peptide is available under UniProt accession number P29460, e.g., which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 39.

[0096] As used herein, an IL-12 variant or mutein refers to any IL-12 molecule that contains at least one amino acid change in at least one of the p35 or p40 subunits as compared to the amino acid sequence of the mature wild-type p35 subunit or the mature wild-type p40 subunit. In some embodiments, the IL-12 variants provided herein can have at least one amino acid mutation in both the p35 and p40 subunits as compared to the amino acid sequence of the mature wild-type p35 or p40.

[0097] In some embodiments, the IL-12 variants provided herein have reduced activity. As used herein, an IL-12 variant with "reduced activity" refers to reduced activity as compared to the activity of the corresponding wild-type IL-12, e.g., wild-type human IL-12. The IL-12 activity can be assessed by any suitable assay known in the art to measure IL-12 activity. For example, IL-12 activity can be assessed by measuring STAT4 phosphorylation (pSTAT4) in cells in response to IL-12 exposure. IL-12 binds to the IL-12 receptor on the surface of T cells, which activates the JAK-STAT signaling pathway in T lymphocytes, and the level of STAT4 phosphorylation is an important indicator of the level of activation of this signaling pathway. In another embodiment, IL-12 activity can be assessed by detecting the level of expression of Ki67. IL-12 binds to the IL-12 receptor on the surface of T cells, which activates the proliferation of T lymphocytes, and the level of Ki67 expression is an important indicator of the proliferation of T lymphocytes.

[0098] In another embodiment, IL-12 activity can be assessed indirectly by measuring the affinity of the IL-12 variant for the IL-12 receptor, e.g., by a biological membrane-based thin layer interferometry assay.

[0099] In some embodiments, the IL-12 variants provided herein have reduced binding to the IL-12 receptor as compared to the binding of wild-type IL-12 to the IL-12 receptor. The IL-12 receptor is a heterodimer containing subunits IL-12Rβ1 (see UniProt ID NO: P42701 for human IL-12Rβ1) and IL-12Rβ2 (see UniProt ID NO: Q99665 for human IL-12Rβ2). In some embodiments, the IL-12 variant has reduced binding affinity to IL-12Rβ1 and / or IL-12Rβ2, e.g., lower than the binding affinity of wild-type IL-12 to its receptor or subunits thereof.

[0100] In some embodiments, the IL-12 variants of the present application comprise a mutation that removes its binding to heparin, and thus it does not bind to heparin.

[0101] In some embodiments, the mutation that removes binding to heparin comprises, e.g., the substitution of the sequence segment "(SKREKKD)" at positions 259-265 of the IL-12 p40 subunit to "(DNTEG)".

[0102] In some embodiments, the IL-12 variants of the present application comprise a mutation that weakens its binding to its receptor, e.g., it comprises a mutation that weakens binding to IL-12Rβ1 and / or IL-12Rβ2.

[0103] In some embodiments, the IL-12 variant of the application comprises a mutation that attenuates binding to its receptor, e.g., IL-12Rβ1, e.g., a mutation (e.g., substitution) of the lysine at position 84 of the IL-12 p40 subunit to another amino acid, e.g., to glutamic acid. Thus, in some embodiments, the IL-12 variant of the application comprises a K84E substitution in the IL-12 p40 subunit variant. In some embodiments, the IL-12 p40 subunit variant comprising a K84E substitution comprises the amino acid sequence set forth in SEQ ID NO: 25, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0104] In some embodiments, the IL-12 variant of the application comprises a K84E substitution and a substitution of the sequence segment “(SKREKKD)” at positions 259-265 to “(DNTEG)” in the IL-12 p40 subunit variant. In some embodiments, the IL-12 p40 subunit variant comprising a K84E substitution and a substitution of the sequence segment “(SKREKKD)” at positions 259-265 to “(DNTEG)” comprises the amino acid sequence set forth in SEQ ID NO: 24, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0105] In some embodiments, the IL-12 variant of the application comprises a K84E substitution and a deletion of amino acids at positions 15-18 in the IL-12 p40 subunit variant. In some embodiments, the IL-12 p40 subunit variant comprising a K84E substitution and a deletion of amino acids at positions 15-18 comprises the amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0106] In some embodiments, the IL-12 variant of the application comprises a mutation that attenuates binding to its receptor, e.g., IL-12Rβ2, e.g., a mutation (e.g., substitution) of the lysine at position 170 of the IL-12 p35 subunit to another amino acid, e.g., to alanine. Thus, in some embodiments, the IL-12 variant of the application comprises a K170A substitution in the IL-12 p35 subunit variant. In some embodiments, the IL-12 p35 subunit variant comprising a K170A substitution comprises the amino acid sequence set forth in SEQ ID NO: 23, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0107] In some embodiments, the IL-12 mutein or IL-12 variant of the application comprises a K84E substitution and a deletion of amino acids at positions 15-18 in the IL-12p40 subunit variant, and a K170A substitution in the IL-12p35 subunit variant. Accordingly, in some embodiments, the IL-12 mutein of the application comprises an IL-12p40 subunit variant and an IL-12p35 subunit variant, wherein the IL-12p40 subunit variant comprises the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; and the IL-12p35 subunit variant comprises the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the IL-12 mutein of the application comprises an IL-12p40 subunit variant and an IL-12p35 subunit variant, wherein the IL-12p40 subunit variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11, and the IL-12p35 subunit variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23.

[0108] In some embodiments, the IL-12 mutein or IL-12 variant of the application comprises a K84E substitution and a substitution of the sequence segment “(SKREKKD)” at positions 259-265 with “(DNTEG)” in the IL-12p40 subunit variant, and a K170A substitution in the IL-12p35 subunit variant. Accordingly, in some embodiments, the IL-12 mutein of the application comprises an IL-12p40 subunit variant and an IL-12p35 subunit variant, wherein the IL-12p40 subunit variant comprises the amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; and the IL-12p35 subunit variant comprises the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the IL-12 mutein of the application comprises an IL-12p40 subunit variant and an IL-12p35 subunit variant, wherein the IL-12p40 subunit variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24, and the IL-12p35 subunit variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23.

[0109] In some embodiments, the IL-12 mutein of the application further comprises a mutation that reduces molecular disulfide bond formation (e.g. for improved drugability), e.g. further comprising a cysteine mutation in the IL-12 p40 subunit, e.g. a C252S substitution.

[0110] In some embodiments, the IL-12 mutein of the application comprises an IL-12 p40 subunit variant and an IL-12 p35 subunit variant linked by a linker, e.g. the C-terminus of the IL-12 p40 subunit variant is linked to the N-terminus of the IL-12 p35 subunit variant by a linker.

[0111] In some embodiments, the linker suitable for linking the IL-12 p40 subunit and the IL-12 p35 subunit is a linker sequence selected from the group consisting of (GS)n, (GSGGS)n(SEQ ID NO: 52), (GGGGS)n(SEQ ID NO: 53), and (GGGS)n(SEQ ID NO: 54), wherein n is an integer of at least 1, 2, 3 or 4. Preferably, the linker comprises (GGGGS)n(SEQ ID NO: 53), wherein n is an integer of at least 1, 2, 3 or 4. n (SEQ ID NO: 55), wherein n is an integer of 1-10, e.g. 1, 2, 3, 4, 5 or 6; more preferably, the linker is GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 42).

[0112] Thus, in some embodiments, the IL-12 mutein comprises

[0113] (i) the amino acid sequence set forth in SEQ ID NO: 40 or 41; or

[0114] (ii) at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 40, and comprises a K84E substitution and a deletion of amino acids at positions 15-18 in the IL-12 p40 subunit variant, and a K170A substitution in the IL-12 p35 subunit variant; or

[0115] (iii) at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 41, and comprises a K84E substitution and a substitution of the sequence segment “(SKREKKD)” at positions 259-265 to “(DNTEG)” in the IL-12 p40 subunit variant, and a K170A substitution in the IL-12 p35 subunit variant.

[0116] II. Immunoconjugates

[0117] In one embodiment of the application, the application relates to an immunoconjugate comprising an IL-12 mutein of the application, and optionally one or more other polypeptides or proteins, such as an antigen binding region that specifically binds to an antigen. In some embodiments, the antigen is an immune checkpoint molecule.

[0118] In one embodiment of the application, the application relates to an immunoconjugate comprising

[0119] (i) an antigen binding region that specifically binds to an immune checkpoint molecule, such as PD-1 ;

[0120] (ii) an IL-12 mutein;

[0121] Optionally the IL-12 mutein is linked to one Fc region, and / or the antigen binding region that specifically binds to PD-1 is linked to another Fc region.

[0122] In some embodiments, the IL-12 mutein is derived from or is a mutein described herein.

[0123] In some embodiments, the antigen binding region that specifically binds to PD-1 suitable for use in the immunoconjugates of the application can comprise or consist of an anti-PD-1 antibody or antigen binding fragment thereof, provided it is capable of specifically binding to PD-1, including but not limited to, for example, a full-length antibody, a half-antibody, a Fab, a Fab', a Fab'-SH, a Fv, a single chain antibody (e.g., scFv), a (Fab')2, a single domain antibody (e.g., VHH), a dAb (domain antibody), a heavy chain antibody, or a linear antibody, etc. that specifically binds to PD-1.

[0124] In some embodiments, the immunoconjugate of the application comprises an Fc dimer.

[0125] In one embodiment, the immunoconjugate can comprise one or more antigen binding regions that specifically bind to PD-1. In one embodiment, the immunoconjugate can comprise one or more IL-12 muteins. In one embodiment, the immunoconjugate comprises 1 antigen binding region that specifically binds to PD-1 and 1 IL-12 mutein. In one embodiment, the immunoconjugate comprises 2 antigen binding regions that specifically bind to PD-1 and 1 IL-12 mutein.

[0126] In one preferred embodiment, the structure of the immunoconjugate of the application is as shown in Figure 1.

[0127] As will be apparent to those skilled in the art, the linker suitable for use in the conjugate molecule of the present application can be any linker known in the art. In some embodiments, the linker can comprise or consist of an immunoglobulin hinge region. The hinge region can be an IgG hinge region, for example, a hinge region of IgGl, IgG2, IgG3, or IgG4. In some embodiments, the linker is a peptide linker. In some embodiments, the linker can comprise an IgGl hinge region, or can comprise a linker sequence selected from the group consisting of (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, wherein n is an integer of at least 1, 2, 3, or 4. Preferably, the linker comprises (G4S) n wherein n is an integer of 1-10, for example, 1, 2, 3, 4, 5, or 6; more preferably, the linker is GGGGSGGGGS (SEQ ID NO: 44) or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 42).

[0128] In some embodiments, the linker can comprise a partial IgG hinge region and a peptide linker (e.g., a glycine linker or a glycine-serine polymer linker). In some embodiments, the linker is DGGGGSGGGGS (SEQ ID NO: 45) or GGGGSGGGGSEPKSS (SEQ ID NO: 22).

[0129] II-1 antigen binding region that specifically binds to PD-1

[0130] In some embodiments, the antigen binding region that specifically binds to PD-1 suitable for use in the immunoconjugate of the present application can comprise or consist of an anti-PD-1 antibody or antigen binding fragment thereof (e.g., a PD-1 antibody disclosed in WO2017024465A1 (incorporated herein in its entirety), for example, Sintilimab), so long as it is capable of specifically binding to PD-1, including but not limited to, for example, a full-length antibody, a half-antibody, a Fab, a Fab’, a Fab’-SH, a Fv, a single chain antibody (e.g., scFv), a (Fab’)2, a single domain antibody such as a VHH, a dAb (domain antibody), a heavy chain antibody, or a linear antibody, etc., that specifically binds to PD-1.

[0131] In some embodiments, the antigen binding region that specifically binds to PD-1 is derived from an antibody that specifically binds to PD-1, for example, a PD-1 antibody disclosed in WO2017024465A1, for example, Sintilimab.

[0132] In some embodiments, the antigen binding region that specifically binds PD-1 comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds PD-1, e.g., a PD-1 antibody disclosed in WO2017024465A1, e.g., Sintilimab.

[0133] In some embodiments, the antigen binding region that specifically binds PD-1 comprises 1, 2, and 3 heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR3, of a known antibody that specifically binds PD-1, e.g., a PD-1 antibody disclosed in WO2017024465A1, e.g., Sintilimab.

[0134] In some embodiments, the antigen binding region that specifically binds PD-1 comprises 1, 2, and 3 light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR3, of a known antibody that specifically binds PD-1, e.g., a PD-1 antibody disclosed in WO2017024465A1, e.g., Sintilimab.

[0135] In some embodiments, the antigen binding region that specifically binds PD-1 comprises 3 heavy chain variable region CDRs and 3 light chain variable region CDRs of a known antibody that specifically binds PD-1, e.g., a PD-1 antibody disclosed in WO2017024465A1, e.g., Sintilimab.

[0136] In some embodiments, the antigen binding region that specifically binds PD-1 comprises a heavy chain variable region and a light chain variable region of a known antibody that specifically binds PD-1, e.g., a PD-1 antibody disclosed in WO2017024465A1, e.g., Sintilimab.

[0137] In some embodiments, the antigen binding region that specifically binds PD-1 comprises a Fab of a known antibody that specifically binds PD-1, e.g., a PD-1 antibody disclosed in WO2017024465A1, e.g., Sintilimab.

[0138] In some embodiments, the antigen binding region that specifically binds PD-1 comprises 3 complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen binding region that specifically binds PD-1 comprises 3 complementarity determining regions (LCDRs) from a light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, the antigen binding region that specifically binds PD-1 comprises 3 complementarity determining regions (HCDRs) from a heavy chain variable region and 3 complementarity determining regions (LCDRs) from a light chain variable region.

[0139] In some aspects, the antigen binding region that specifically binds PD-1 comprises a heavy chain variable region (VH). In some aspects, the antigen binding region that specifically binds PD-1 comprises a light chain variable region (VL). In some aspects, the antigen binding region that specifically binds PD-1 comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises 3 complementarity determining regions (CDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises 3 complementarity determining regions (CDRs) from a light chain variable region, LCDR1, LCDR2, and LCDR3.

[0140] In some embodiments, the heavy chain variable region of the antigen binding region that specifically binds PD-1

[0141] (i) comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 29; or

[0142] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 29; or

[0143] (iii) comprises or consists of an amino acid sequence that has 1 or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 29, preferably the amino acid alterations do not occur in the CDR regions.

[0144] In some embodiments, the light chain variable region of the antigen binding region that specifically binds PD-1

[0145] (i) comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 33; or

[0146] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 33; or

[0147] (iii) consists of an amino acid sequence which has 1 or more, preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1, amino acid alterations, preferably amino acid substitutions, more preferably amino acid conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 33, preferably said amino acid alterations do not occur in CDR regions.

[0148] In some embodiments, the three complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2 and HCDR3, of an antigen binding region that specifically binds to PD-1 are selected from

[0149] (i) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as shown in SEQ ID NO: 29, or

[0150] (ii) a sequence which comprises, in total, over the three HCDR regions, at least one and not more than 5, 4, 3, 2, or 1 amino acid alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the sequence of any of (i),

[0151] wherein said HCDR1, 2 and 3 can each be determined according to any CDR-determining scheme, e.g. according to the scheme of Kabat, AbM, Chothia, Contact or IMGT, respectively, or a combination thereof;

[0152] e.g. said HCDR1 is determined by the AbM scheme, said HCDR2, HCDR3 are determined by the Kabat scheme.

[0153] In some embodiments, the three complementarity determining regions (LCDRs) from a light chain variable region, LCDR1, LCDR2 and LCDR3, of an antigen binding region that specifically binds to PD-1 are selected from

[0154] (i) the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as shown in SEQ ID NO: 33, or

[0155] (ii) a sequence which comprises, in total, over the three LCDR regions, at least one and not more than 5, 4, 3, 2, or 1 amino acid alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the sequence of any of (i),

[0156] wherein said LCDR1, 2 and 3 can each be determined according to any CDR-determining scheme, e.g. according to the scheme of Kabat, AbM, Chothia, Contact or IMGT, respectively, or a combination thereof;

[0157] For example, the LCDR1, 2 and 3 are determined by the Kabat scheme, respectively.

[0158] In some embodiments, the antigen binding region that specifically binds to PD-1 comprises 3 heavy chain variable region contained complementarity determining regions (HCDRs) consisting of the amino acid sequence of SEQ ID NO: 30, 31 and 32 and 3 light chain variable region contained complementarity determining regions (LCDRs) consisting of the amino acid sequence of SEQ ID NO: 34, 35 and 36.

[0159] In some embodiments, the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 30, or the HCDR1 comprises an amino acid sequence having one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 30.

[0160] In some embodiments, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 31, or the HCDR2 comprises an amino acid sequence having one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 31.

[0161] In some embodiments, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 32, or the HCDR3 comprises an amino acid sequence having one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 32.

[0162] In some embodiments, the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 34, or the LCDR1 comprises an amino acid sequence having one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 34.

[0163] In some embodiments, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 35, or the LCDR2 comprises an amino acid sequence having one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 35.

[0164] In some embodiments, the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 36, or the LCDR3 comprises an amino acid sequence having one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 36.

[0165] In some specific embodiments of the application, the antigen binding region that specifically binds to PD-1 comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2 and / or a LCDR3 as described above. In some specific embodiments of the application, the antigen binding region that specifically binds to PD-1 comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2 and a LCDR3 as described above.

[0166] In some specific embodiments of the application, the antigen binding region that specifically binds to PD-1 comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2 and a LCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 30; the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 31 ; the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 32; the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 34; the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 35; and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 36.

[0167] In some specific embodiments of the application, the antigen binding region that specifically binds to PD-1 comprises a HCDR1 as set forth in SEQ ID NO: 30, a HCDR2 as set forth in SEQ ID NO: 31, a HCDR3 as set forth in SEQ ID NO: 32; a LCDR1 as set forth in SEQ ID NO: 34, a LCDR2 as set forth in SEQ ID NO: 35 and a LCDR3 as set forth in SEQ ID NO: 36.

[0168] In some specific embodiments of the application, the antigen binding region that specifically binds to PD-1 comprises a VH and a VL, wherein

[0169] the VH comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 29 or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the VL comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 33 or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0170] In some embodiments of the application, the antigen binding region that specifically binds to PD-1 comprises a VH and a VL, wherein the VH and VL comprise or consist of the amino acid sequences as set forth in SEQ ID NO: 29 and SEQ ID NO: 33, respectively.

[0171] In some embodiments of the application, the antigen binding region that specifically binds to PD-1 is a Fab against PD-1.

[0172] II-2 Fab fragment

[0173] In some embodiments, the antigen binding region that specifically binds to PD-1 of the application is a Fab fragment. A Fab fragment suitable for use as the antigen binding region that specifically binds to PD-1 consists of two polypeptide chains comprising an antibody VH, CH1, VL and CL domain, wherein the VH pairs with the VL and the CH1 pairs with the CL to form the antigen binding region. In some embodiments, in a Fab, one chain comprises or consists of, from N- to C-terminus, a VH and a CH1 (i.e. VH-CH1), and the other chain comprises or consists of, from N- to C-terminus, a VL and a CL (i.e. VL-CL). In some embodiments, in the immunoconjugate of the application, the Fab can be linked at the N-terminus of the Fc domain of the antibody via the C-terminus of the chain comprising the VH. Preferably, the Fab comprises a VH-CH1 chain and a VL-CL chain, and can be linked to the antibody Fc domain via the C-terminus of the CH1 of the VH-CH1 chain. In some embodiments, the linkage is a direct linkage, or via a linker. In this context, the Fab chain comprising or consisting of a VH-CH1 is also referred to as Fab heavy chain, and the Fab chain comprising or consisting of a VL-CL is also referred to as Fab light chain.

[0174] In some embodiments, the CH1 is or is from a CH1 of (human) IgGl, IgG2, IgG3 or IgG4, preferably a CH1 of IgGl. In some embodiments, the CH1 is or is from a CH1 of IgGl, IgG2, IgG3 or IgG4, preferably a CH1 of IgGl, which has one or more amino acid substitutions compared to the wild type CH1 of IgGl, IgG2, IgG3 or IgG4, respectively. In some embodiments, the CH1 is or is from a CH1 of IgGl, IgG2, IgG3 or IgG4, preferably a CH1 of IgGl, which has one or more amino acid substitutions compared to the wild type CH1 of IgGl, IgG2, IgG3 or IgG4, respectively, and which does not affect the ability of the CH1 to pair with a CL.

[0175] (i) comprises or consists of an amino acid sequence which has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 28;

[0176] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 28; or

[0177] (iii) comprises or consists of an amino acid sequence which has 1 or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 28.

[0178] In some embodiments, the light chain constant region CL is or is from a (human) Kappa light chain constant region or a Lambda light chain constant region. In some embodiments, the CL is a Kappa light chain constant region. In some embodiments, the CL is a Lambda light chain constant region.

[0179] (i) comprises or consists of an amino acid sequence which has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 37;

[0180] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 37; or

[0181] (iii) comprises or consists of an amino acid sequence which has 1 or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 37.

[0182] In some embodiments, the antigen binding region of the application that specifically binds to PD-1 is a Fab that specifically binds to PD-1 comprising a heavy chain variable region VH and a light chain variable region VL of an antigen binding region that specifically binds to PD-1 as defined herein. In some embodiments, the Fab heavy chain of the Fab that is the antigen binding region that specifically binds to PD-1 comprises or consists of a VH and a CH1, wherein the VH is a VH of an antigen binding region that specifically binds to PD-1 as described herein. In some embodiments, the Fab light chain of the Fab that is the antigen binding region that specifically binds to PD-1 comprises or consists of a VL and a CL, wherein the VL is a VL of an antigen binding region that specifically binds to PD-1 as defined herein. In some embodiments, the antigen binding region that specifically binds to PD-1 comprises a CH1 from IgG1, and / or a Kappa light chain constant region.

[0183] In some embodiments, the Fab heavy chain that specifically binds to PD-1

[0184] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 43;

[0185] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 43; or

[0186] (iii) comprises or consists of an amino acid sequence that has 1 or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1 ) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 43.

[0187] In some embodiments, the Fab light chain that specifically binds to PD-1

[0188] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 14;

[0189] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 14; or

[0190] (iii) an amino acid sequence which comprises or consists of 1 or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 14.

[0191] In some embodiments, the Fab specifically binding PD-1 comprises or consists of a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 43, and the Fab light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14.

[0192] II-3 Fc region

[0193] In some embodiments, the immunoconjugate of the application further comprises an Fc region, for example it comprises a first Fc region and a second Fc region, wherein the first and second Fc region can be the same or different.

[0194] In some embodiments, the first and second Fc region are different and are capable of dimerizing to form a heterodimeric Fc scaffold.

[0195] In the present context, Fc region refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region, and can include native sequence Fc regions and variant Fc regions. Native sequence Fc regions encompass the naturally occurring sequences of the various immunoglobulin Fc sequences, for example, the Fc regions of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi: 10.3389 / fimmu.2014.00520). In some embodiments, the Fc region of the application comprises antibody CH2 and CH3. In some embodiments, the antibody Fc region can also carry at the N-terminus an IgG hinge region or a partial IgG hinge region, for example, an IgGl hinge region or a partial IgGl hinge region, for example, the sequence D221 to P230 according to EU numbering. Mutations can be contained in the hinge region.

[0196] Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region is in accordance with the EU numbering system, also referred to as the EU index, as described in Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0197] In some embodiments, the Fc region is or is from a human IgG Fc, e.g., a human IgGl Fc, a human IgG2 Fc, a human IgG3 Fc, or a human IgG4 Fc. In one embodiment, the Fc region comprises or consists of the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence having at least 90% identity, e.g., 95%, 96%, 97%, 98%, 99% or more, thereto.

[0198] As will be appreciated by those skilled in the art, to facilitate the formation of the immunconjugate or fusion protein comprising the IL-12 mutein of the application as a heterodimer, the Fc region comprised by the immunconjugate of the application can comprise mutations that favor heterodimerization of the first Fc region with the second Fc region. In one embodiment, mutations are introduced in the CH3 region of both Fc regions.

[0199] Methods to facilitate Fc region heterodimerization are known in the art. For example, the CH3 region of the first Fc region and the CH3 region of the second Fc region are engineered in a complementary fashion such that each CH3 region (or the heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with the other CH3 region of complementary engineering (such that the CH3 regions of the first and second Fc region heterodimerize and no homodimers are formed between two first CH3 regions or two second CH3 regions).

[0200] Preferably, based on the Knob-into-hole technology, corresponding Knob mutations and Hole mutations are introduced in the first Fc region and the second Fc region. This technology is described, e.g., in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). In some embodiments, the knob mutations and hole mutations can further comprise cysteine mutations and thus introduce disulfide bonds. The disulfide bonds can be used to stabilize the final immunconjugate.

[0201] In a particular embodiment, in the CH3 region of one Fc region the threonine residue at position 366 is replaced with a tryptophan residue (T366W) (knob mutation) and in the CH3 region of the other Fc region the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (hole mutation), optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (numbering in accordance with EU index).

[0202] In some embodiments, the Fc region can further comprise a cysteine residue substitution to obtain a non-native disulfide bond linkage. In some embodiments, in one Fc region the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C), in particular the serine residue at position 354 is replaced with a cysteine residue, and in the other Fc region the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C) (numbering in accordance with EU index). Thus, in a further embodiment, in the CH3 region of one Fc region the threonine residue at position 366 is replaced with a tryptophan residue (T366W) and the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C), in particular the serine residue at position 354 is replaced with a cysteine residue, and in the CH3 region of the other Fc region the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (hole mutation), optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering in accordance with EU index), optionally the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C) (numbering in accordance with EU index).

[0203] In a particular embodiment, one Fc region comprises the amino acid substitution T366W and the other Fc region comprises the amino acid substitutions T366S, L368A and Y407V (numbering in accordance with EU index).

[0204] In a particular embodiment, one Fc region comprises the amino acid substitutions S354C and T366W and the other Fc region comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering in accordance with EU index).

[0205] Mutations can also be introduced in the first and second Fc regions based on the Innobody technology. This technology is described in, for example, PCT / CN2021 / 143141, the disclosure of which is hereby incorporated by reference in its entirety.

[0206] The Fc region in the antibodies of the application can also be mutated to obtain desired properties. Mutations to the Fc region are known in the art.

[0207] In one embodiment, the Fc region is modified in the properties of the effector function of the Fc region, e.g., the complement-activating function of the Fc region. In one embodiment, the effector function has been reduced or eliminated relative to the wild-type isotype Fc region. In one embodiment, the effector function is reduced or eliminated by a method selected from the group consisting of: use of an Fc isotype that naturally has reduced or eliminated effector function, and Fc region modification.

[0208] In a preferred embodiment, the Fc region has reduced effector function mediated by the Fc region, e.g., reduced or eliminated ADCC or ADCP or CDC effector function, e.g., comprising mutations that effect the above functions. As will be appreciated by those skilled in the art, depending on the intended use of the binding molecule, e.g., antibody molecule, of the application, the binding molecule, e.g., antibody molecule, of the application can also comprise modifications in the Fc domain that alter the binding affinity for one or more Fc receptors. In one embodiment, the Fc receptor is an Fcy receptor, particularly a human Fcy receptor. In some embodiments, the Fc region comprises mutations that reduce binding to Fcy receptors. For example, in some embodiments, the Fc region used in the application has mutations that reduce binding to Fcy receptors, e.g., L234A / L235A mutations. In yet another preferred embodiment, the Fc fragment can have mutations that result in increased serum half-life, e.g., mutations that improve the binding of the Fc fragment to FcRn. In some embodiments, the Fc fragment comprises the amino acid sequence set forth in SEQ ID NO: 27, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 27 and comprises L234A / L235A mutations.

[0209] Thus, in a particular embodiment, the immunoconjugate of the application comprises two Fc region heterodimerization, wherein one Fc region polypeptide comprises the knob mutations S354C and T366W, and the other Fc region polypeptide comprises the hole mutations Y349C, T366S, L368A, and Y407V.

[0210] Thus, in a particular embodiment, the immunoconjugate of the application comprises two Fc regions that are heterodimerized, wherein one Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 and the other Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2.

[0211] Thus, in a particular embodiment, the immunoconjugate of the application comprises two Fc regions that are heterodimerized, wherein one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 1 and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 2.

[0212] Thus, in a particular embodiment, the immunoconjugate of the application comprises two Fc regions that are heterodimerized, wherein one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 1 and comprises the mutations S354C and T366W, and optionally L234A / L235A mutations, and the other Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 2 and comprises the mutations Y349C, T366S, L368A and Y407V, and optionally L234A / L235A mutations.

[0213] In some embodiments, the Fc region further comprises other mutations that facilitate heterodimer purification, such as the mutations disclosed in WO2022143912A1, which is incorporated herein in its entirety.

[0214] II-4 Exemplary immunoconjugate structures and immunoconjugates

[0215] In one embodiment, the immunoconjugate of the application specifically binds PD-1 and IL-12 receptor and comprises an antigen binding region that specifically binds PD-1 as defined herein, and an IL-12 mutein that specifically binds IL-12 receptor as defined herein.

[0216] In some embodiments, the antigen binding region that specifically binds PD-1 is a Fab fragment as defined herein.

[0217] Thus, in some embodiments, the immunoconjugate of the application comprises

[0218] 1 Fab fragment specifically binding PD-1 as defined herein and 1 IL-12 mutein specifically binding IL-12 receptor as defined herein; optionally, the Fab heavy chain of the Fab fragment is linked to a first Fc region and the IL-12 mutein is linked to a second Fc region, optionally, the first and second Fc region comprise mutations promoting Fc region heterodimerization, e.g. Knob-into-hole mutations.

[0219] In a particular embodiment, the IL-12 mutein is linked to the Fc region via a linker at the C-terminus of the IL-12 p35 subunit.

[0220] In a particular embodiment, the Fab fragment specifically binding PD-1 is linked to the Fc region via the C-terminus of the Fab heavy chain.

[0221] In a particular embodiment, the 2 Fab fragments specifically binding PD-1 can be identical or different, e.g. they have identical Fab heavy and light chains, or they have different Fab heavy and / or Fab light chains.

[0222] In a particular embodiment, the Fab heavy chains of the 2 Fab fragments specifically binding PD-1 are linked via a linker, e.g. the C-terminus of the first Fab heavy chain is linked to the N-terminus of the second Fab heavy chain via a linker. In some embodiments, the linker linking the two Fab heavy chains comprises or is D (e.g. at the N-terminus) and a glycine-serine polymer such as (GS) n In some embodiments, the linker linking the two Fab heavy chains comprises or is D (GGGGS) n (SEQ ID NO: 56), wherein n is an integer from 1 to 10, e.g. n = 1, 2, 3 or 4.

[0223] In some embodiments, the linker linking the two Fab heavy chains comprises or consists of the amino acid sequence set forth in SEQ ID NO: 44 or 45.

[0224] In some embodiments, the linker linking the IL-12 p40 and IL-12 p35 subunit variants in the IL-12 mutein comprises a glycine-serine polymer, e.g. (GGGGS) n wherein n is an integer from 1 to 10, e.g. n = 1, 2, 3, 4, 5 or 6. In some embodiments, the linker linking the IL-12 p40 and IL-12 p35 subunit variants in the IL-12 mutein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 42.

[0225] In some embodiments, the linker connecting the IL-12 mutein to the Fc region comprises a glycine-serine polymer such as (GS) n In some embodiments, the linker connecting the IL-12 mutein to the Fc region further comprises a partial hinge region (preferably a partial hinge region with a C220S mutation), for example EPKSS (SEQ ID NO: 57). In some embodiments, the linker connecting the IL-12 mutein to the Fc region comprises (GS) n EPKSS (SEQ ID NO: 58), wherein n is an integer from 1-10, for example n = 1, 2, 3, or 4. In some embodiments, the linker connecting the IL-12 mutein to the Fc region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22.

[0226] Thus, in some embodiments, the immunoconjugate of the application comprises

[0227] a first heavy chain: a Fab heavy chain that specifically binds PD-1 - a first Fc region;

[0228] a light chain: a Fab light chain that specifically binds PD-1; and

[0229] a second heavy chain: an IL-12 mutein - a linker 1 - a second Fc region;

[0230] wherein the Fab heavy chain and the Fab light chain form a Fab fragment that specifically binds PD-1 (as defined in Section II-2);

[0231] the IL-12 mutein is an IL-12 mutein as defined in Section I, for example it comprises an IL-12p40 subunit variant and an IL-12p35 subunit variant connected via a linker 2, for example IL-12p40 subunit variant - linker 2 - IL-12p35 subunit variant, in some embodiments the IL-12p40 subunit variant is connected at its C-terminus to the N-terminus of the IL-12p35 subunit variant via the linker 2 (C-terminus of the IL-12p40 subunit variant - linker 2 - N-terminus of the IL-12p35 subunit variant);

[0232] optionally the first Fc region and the second Fc region are Fc regions as defined in Section II-3, for example the first Fc region comprises hole mutations such as Y349C / T366S / L368A / Y407V; and / or the second Fc region comprises knob mutations such as S354C and T366W;

[0233] optionally the first Fc region and / or the second Fc region further comprises mutations that reduce binding to Fc gamma receptors, for example L234A / L235A mutations;

[0234] Optionally linker 1 is an amino acid sequence as set forth in SEQ ID NO: 22, and / or linker 2 is an amino acid sequence as set forth in SEQ ID NO: 42.

[0235] Thus, in some embodiments, the immunoconjugate of the application comprises a first heavy chain, a second heavy chain and a light chain as defined above, wherein

[0236] the first heavy chain comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO: 12, or an amino acid sequence which has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto;

[0237] the second heavy chain comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO: 17 or 18, or an amino acid sequence which has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto; and / or

[0238] the light chain comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO: 14, or an amino acid sequence which has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto.

[0239] In some embodiments, the immunoconjugate of the application comprises a first heavy chain, a second heavy chain and a light chain as defined above, wherein

[0240] the first heavy chain comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO: 12;

[0241] the second heavy chain comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO: 17 or 18; and

[0242] the light chain comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO: 14.

[0243] Thus, in some embodiments, the immunoconjugate of the application comprises

[0244] a first heavy chain: Fab heavy chain 1 that specifically binds PD-1 - linker 3 - Fab heavy chain 2 that specifically binds PD-1 - first Fc region;

[0245] a first light chain: Fab light chain 1 that specifically binds PD-1 ;

[0246] a second light chain: Fab light chain 2 that specifically binds PD-1 ;

[0247] a second heavy chain: IL-12 mutein - linker 1 - second Fc region;

[0248] wherein the Fab heavy chain 1 and the Fab light chain 1 form a first Fab fragment that specifically binds to PD-1 (as defined in section II-2);

[0249] the Fab heavy chain 2 and the Fab light chain 2 form a second Fab fragment that specifically binds to PD-1 (as defined in section II-2);

[0250] wherein the IL-12 mutein is an IL-12 mutein as defined in section I, e.g. comprising an IL-12 p40 subunit variant and an IL-12 p35 subunit variant connected via linker 2, e.g. IL-12 p40 subunit variant-linker 2-IL-12 p35 subunit variant; in some embodiments, the IL-12 p40 subunit variant is connected at its C-terminus via linker 2 to the N-terminus of the IL-12 p35 subunit variant (C-terminus of IL-12 p40 subunit variant-linker 2-N-terminus of IL-12 p35 subunit variant);

[0251] optionally, the first Fc region and the second Fc region are Fc regions as defined in section II-3, e.g. the first Fc region comprises hole mutations, e.g. Y349C / T366S / L368A / Y407V; and / or the second Fc region comprises knob mutations, e.g. S354C and T366W;

[0252] optionally the first Fc region and / or the second Fc region further comprises mutations having reduced binding to Fc gamma receptors, e.g. L234A / L235A mutations;

[0253] optionally linker 1 is an amino acid sequence as set forth in SEQ ID NO: 22, linker 2 is an amino acid sequence as set forth in SEQ ID NO: 42, and / or linker 3 is an amino acid sequence as set forth in SEQ ID NO: 44 or 45;

[0254] wherein the first Fab fragment is the same or different from the second Fab fragment, preferably the same.

[0255] Thus, in some embodiments, the immunoconjugate of the application comprises a first heavy chain, a second heavy chain, a first light chain and a second light chain as defined above, wherein

[0256] the first heavy chain comprises, consists of or consists essentially of an amino acid sequence as set forth in SEQ ID NO: 13, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto;

[0257] the second heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 17 or 18 or 20 or 21, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, or consisting of said amino acid sequence; and / or

[0258] the first and second light chains each comprise an amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, or consisting of said amino acid sequence.

[0259] In some embodiments, the immunoconjugate of the application comprises a first heavy chain, a second heavy chain, a first light chain and a second light chain as defined above, wherein

[0260] the first heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 13, or consists of said amino acid sequence;

[0261] the second heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 17 or 18 or 20 or 21, or consists of said amino acid sequence; and

[0262] the first and second light chains each comprise an amino acid sequence set forth in SEQ ID NO: 14, or consists of said amino acid sequence.

[0263] III. Polynucleotides, Vectors and Hosts

[0264] The present application provides nucleic acids encoding any of the chains or any monomer or domain of the IL-12 muteins of the application or the immunoconjugates of the application. The polynucleotide sequences encoding each chain can be generated using methods well known in the art. In addition, the polynucleotides and nucleic acids of the application can comprise a segment encoding a secretion signal peptide and operably linked to the coding sequence of the antibody or protein in the immunoconjugates of the application, such that the secretion of the immunoconjugates of the application and each of its chains can be directed.

[0265] The present application also provides vectors comprising the nucleic acids of the application. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. The vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage or yeast artificial chromosomes (YAC). In a preferred embodiment, the expression vector of the application is a pcDNA vector, such as a pcDNA3.1 expression vector.

[0266] The present application also provides host cells comprising the nucleic acid or the vector. Host cells suitable for replication and support of expression of the IL-12 muteins of the present application or the immunoconjugates of the present application are well known in the art. Such cells can be transfected or transduced with the particular expression vector and large quantities of the vector-containing cells can be grown for seeding large scale fermentors to obtain sufficient quantities of the immunoconjugates for clinical use.

[0267] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell (e.g., a CHO cell or a 293 cell, e.g., an Expi293 cell). Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (WI-38), human hepatoma cells (Hep G2), mouse mammary tumor cells (MMT060562), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr- CHO cells, and myeloma cell lines, such as YB2 / 0, NS0, P3X63 and Sp2 / 0.

[0268] In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphocyte (e.g., YB2 / 0, NS0, Sp2 / 0 cell).

[0269] IV. Methods of manufacture

[0270] In another aspect, the present application provides a method of making an IL-12 mutein of the present application or an immunoconjugate of the present application, the method comprising culturing a host cell comprising a nucleic acid encoding the IL-12 mutein or each chain of the immunoconjugate, as provided above, under conditions suitable for expression of the IL-12 mutein, or each chain of the immunoconjugate, and optionally recovering the IL-12 mutein or immunoconjugate from the host cell (or host cell culture medium).

[0271] In one embodiment, a vector comprising nucleic acid encoding each chain of the IL-12 mutein or the immunoconjugate of the application is introduced into a cell for expression, and the cell (or cell culture supernatant) is subsequently collected, the IL-12 mutein or immunoconjugate is extracted, and purified to obtain the immunoconjugate. In a particular embodiment, the purification method is an affinity purification method (e.g., Fc tag protein purification). In another particular embodiment, the purification method is ion exchange purification (e.g., anion and / or cation purification).

[0272] V. Pharmaceutical compositions and pharmaceutical formulations

[0273] The present application also includes compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising an IL-12 mutein or immunoconjugate, and compositions comprising a polynucleotide encoding an IL-12 mutein or immunoconjugate. These compositions can also optionally comprise suitable pharmaceutical adjuvants, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, as known in the art.

[0274] Pharmaceutical compositions or pharmaceutical formulations comprising the immunoconjugate of the application can be prepared by conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions can be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0275] The compositions, e.g., pharmaceutical compositions or pharmaceutical formulations, can also comprise other active ingredients, such as one or more other therapeutic agents (e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, etc.).

[0276] In some embodiments, the compositions are used for the prevention or treatment of cancer.

[0277] VI. Combination products

[0278] In one aspect, the present application also provides combinations (e.g., pharmaceutical combinations) comprising an IL-12 mutein or immunoconjugate of the application, and one or more other therapeutic agents. The combination products of the application can be used in the therapeutic methods or uses of the application.

[0279] The present application also provides kits comprising the combination, e.g., the kits comprise in the same package:

[0280] - a first container containing an IL-12 mutein or immunoconjugate of the application or a pharmaceutical comprising the IL-12 mutein or immunoconjugate;

[0281] - a second container comprising a pharmaceutical composition of one or more other therapeutic agents.

[0282] In some embodiments, the combination product or kit is for use in the prevention or treatment of a tumor, e.g., a cancer.

[0283] In some embodiments, when the IL-12 muteins or immunoconjugates of the application are used to treat a tumor, the other therapeutic agents that can be administered in combination or combination with the molecules of the application encompass various therapeutic agents used to treat a tumor, e.g., chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents (e.g., immune checkpoint inhibitors or agonists). Exemplary other antibodies include antibodies that specifically bind to an immune checkpoint.

[0284] VII. Methods of treatment and uses

[0285] In one aspect, the application provides a method of preventing or treating a disease in a subject, comprising administering to the subject an effective amount of an IL-12 mutein or immunoconjugate, a pharmaceutical composition, a pharmaceutical combination, or a kit of the application. In some embodiments, the disease is a PD-1 -associated disease and / or disorder. In some embodiments, the disease is, e.g., a tumor such as a cancer.

[0286] In some embodiments, the IL-12 muteins or immunoconjugates of the application can be used to stimulate the immune system of a host, e.g., to enhance the immune response of a cell. "Stimulating the immune system" in accordance with any of the above embodiments can include any one or more of an overall increase in immune function, an increase in T cell function, an increase in B cell function, a restoration of lymphocyte function, an increase in IL-12 receptor expression, an increase in T cell responsiveness, an increase in T cell activity or natural killer cell activity or lymphokine-activated killer cell (LAK) activity, an increase in T cell or natural killer cell survival, an increase in expression of cell-killing effector proteins, and the like.

[0287] In some embodiments, the application relates to an IL-12 mutein or immunoconjugate, a pharmaceutical composition, a pharmaceutical combination, or a kit of the application for use in therapy, e.g., for use in the treatment of a PD-1 -associated disease and / or disorder.

[0288] In some embodiments, the application relates to a method of treating a disease, e.g., a disease or disorder mentioned herein, using an IL-12 mutein or immunoconjugate, a pharmaceutical composition, a pharmaceutical combination, or a kit of the application, or to the use for such a treatment, or to the use for the manufacture of a medicament for such a treatment.

[0289] In some embodiments, the disease is a PD-1 related disease and / or disorder. In some embodiments, the disease is e.g. a tumor such as a cancer. In some embodiments, the tumor is a solid tumor or a hematological tumor and a metastatic lesion. In one embodiment, examples of solid tumors include malignant tumors. The cancer can be in an early, intermediate or advanced stage or be a metastatic cancer. In some embodiments, the tumor encompasses tumor immune escape.

[0290] In some embodiments, the tumor is a PD-1 positive tumor or cancer. In some embodiments, the tumor is associated with abnormal expression or abnormal activity of PD-1.

[0291] In some embodiments, a PD-1 positive tumor or cancer refers to abnormal expression or activity of PD-1 in a subject having said tumor cancer. In some embodiments, the subject (in particular an adult subject) has PD-1 expression. In some embodiments, the subject has (e.g. elevated levels of, e.g. nucleic acid or protein levels or activity of) PD-1 in immune cells, e.g. activated T cells (e.g. compared to a healthy subject).

[0292] In some embodiments, the subject also has (e.g. elevated levels of, e.g. nucleic acid or protein levels or activity of) PD-1 in a biological sample (e.g. tumor tissue, e.g. tumor tissue microenvironment) of the subject (e.g. compared to a biological sample of a healthy subject (e.g. corresponding tissue or immune cells in a healthy subject), or compared to PD-1 in a proximal healthy tissue or immune cells of the subject).

[0293] PD-1 positive tumor or cancer also encompasses tumors or cancers with abnormal activity of PD-1 due to abnormal expression or activity of PD-L1 and / or PD-L2. Thus in some embodiments, the tumor or cancer is a tumor or cancer characterized by elevated protein levels and / or nucleic acid levels (e.g. elevated expression) of PD-L1 and / or PD-L2, e.g. the tumor cells of the cancer have elevated protein levels and / or nucleic acid levels (e.g. elevated expression) of PD-L1 and / or PD-L2, e.g. compared to cells of a corresponding tissue of a healthy subject, or compared to proximal healthy cells of the tumor cells.

[0294] In some embodiments, a PD-1 positive tumor or cancer refers to immune cells, e.g., activated T cells, having elevated expression of PD-1 in individuals having all tumor cancers. In some embodiments, the individual has intermediate or high expression of PD-1 in immune cells. In some embodiments, a PD-1 positive tumor refers to abnormal expression of PD-1 in immune cells. In some embodiments, abnormal expression of PD-1 refers to expression of PD-1 on immune cells that is higher than PD-1 expression in control cells, e.g., healthy immune cells of a healthy individual.

[0295] In a particular embodiment, the IL-12 mutein or immunoconjugate molecules of the application are capable of killing tumor cells, and / or inhibiting tumor cell proliferation, e.g., tumor cells expressing PD-L1 and / or PD-L2.

[0296] In some embodiments, the tumor is a tumor that has been treated with other therapies, e.g., chemotherapy and / or radiation therapy. In some embodiments, the tumor is a tumor or cancer that is resistant to known drugs, e.g., known anti-PD-1 antibodies, e.g., a refractory tumor or cancer.

[0297] In some embodiments, the tumor or cancer is selected from intestinal cancer, breast cancer, lung cancer, melanoma, or pancreatic cancer.

[0298] Depending on its therapeutic use, the IL-12 mutein or immunoconjugate of the application can also be administered in combination with one or more other therapies, e.g., other treatment modalities and / or other therapeutic agents, for the uses described herein, e.g., for the prevention and / or treatment of the relevant diseases or conditions mentioned herein.

[0299] In some embodiments, when the IL-12 mutein or immunoconjugate of the application is used to treat a tumor, the treatment modalities include surgery, radiation therapy, local or focused irradiation, etc.

[0300] In other aspects, the application provides the use of the IL-12 mutein or immunoconjugate of the application, or a composition or combination product comprising the same, for the manufacture or preparation of a medicament for the uses described herein, e.g., for the prevention or treatment of the relevant diseases or conditions mentioned herein.

[0301] In other aspects, the application also provides the IL-12 mutein or immunoconjugate of the application, or a composition or medicament or formulation or combination product comprising the same, for use in therapy, e.g., for use in the treatment of the relevant diseases or conditions mentioned herein.

[0302] The subject can be a mammal, for example, a primate, preferably, a higher primate, e.g., a human (e.g., an individual having or at risk of having a disease described herein). In one embodiment, the subject has a disease described herein (e.g., a cancer) or is at risk of having a disease described herein. In some embodiments, the subject receives or has received other therapy, e.g., chemotherapy treatment and / or radiation therapy. In some embodiments, the subject has previously received or is receiving immunotherapy.

[0303] Combination therapies of the application encompass combined administration (e.g., two or more therapeutic agents are included in the same formulation or in separate formulations), and separate administration, in which case, administration of the IL-12 mutein or immunoconjugate of the application or composition or medicament or formulation comprising the same can precede, follow, or be concurrent with the administration of the other therapeutic agent and / or agents.

[0304] The route of administration of the pharmaceutical composition is in accordance with known methods, e.g., orally, by intravenous injection, intraperitoneally, intracerebrally (intraparenchymal), intracerebroventricularly, intramuscularly, intraocularly, intraarterially, intraportally, or intralesionally; by sustained release systems or by implantation devices. In certain embodiments, the composition can be administered by bolus injection or by continuous infusion or by implantation devices.

[0305] The composition can also be administered topically via an implanted membrane, sponge, or another suitable material on which the desired molecules are absorbed or encapsulated. In certain embodiments, when an implantation device is used, the device can be implanted into any suitable tissue or organ, and can deliver the desired molecules via diffusion, timed release bolus, or continuous administration.

[0306] In some embodiments, when the IL-12 mutein or immunoconjugate of the application is used to treat a tumor, the other therapeutic agent that can be administered in combination or in combination with the molecule of the application encompasses various therapeutic agents used to treat a tumor, e.g., a chemotherapeutic agent, an angiogenesis inhibitor, a cytokine, a cytotoxic agent, another antibody, a small molecule drug, or an immunomodulatory agent (e.g., an immune checkpoint inhibitor or agonist). Exemplary other antibodies include antibodies that specifically bind an immune checkpoint.

[0307] Any literature, including patents, patent applications, and publications, cited herein is incorporated by reference in its entirety.

[0308] Any or all of the features discussed above in connection with the present application can be combined in various embodiments of the present application. Any embodiment described above in connection with the present application can be combined.

[0309] The following examples further illustrate the application, however, it is understood that the examples are described by way of illustration and not by way of limitation, and that modifications can be made by those skilled in the art without departing from the scope of the application.

[0310] Example:

[0311] Example 1. Design of anti-hPD-1 and IL-12 mutein immunoconjugate

[0312] One of the designs of the present application is a molecule that can bind to human PD-1 to block the binding of PD-1 to PD-L1, release the immune brake mechanism, and also bind to IL-12 receptors on T cells or NK cells to activate T cells, enhancing the effect of PD-1 antibody immunotherapy. The molecular form is shown in Figure 1, which includes two parts: 1) the antibody sequence that binds to PD-1 is derived from WO2017024465A1; 2) IL-12 mutein (IL-12m) as shown in Table 1.

[0313] Table 1. IL-12 mutein

[0314] Table 2. Design of IL-12 immunoconjugate

[0315] Example 2. Preparation of IL-12 immunoconjugate

[0316] Vector construction

[0317] IL-12 immunoconjugate is based on Knob-into-hole Fc heterodimer, IL-12 mutein sequence is constructed to the N-terminal of Fc-Knob (SEQ ID NO: 1), and the sequence of anti-PD-1 is constructed to the N-terminal of Fc-Hole (SEQ ID NO: 2), respectively, into the pcDNA3.1 vector, and then co-expressed in cells.

[0318] The vector containing the gene encoding the fusion protein was transfected into HEK293 cells for expression using the method of transient transfection. First, prepare the plasmid DNA and transfection reagent PEI in the clean bench, take 3 ml of Opti-MEM medium (Gibco, 31985-070) into a 50 ml centrifuge tube, add 30 μg of DNA corresponding to the plasmid, filter the Opti-MEM medium containing the plasmid with a 0.22 μm filter, then add 90 μg of PEI (1 g / L), and stand for 20 min. Gently pour the DNA / PEI mixture into 27 ml of HEK293 cells and mix well, continue to culture at 37°C, 8% CO2 for 6 days.

[0319] Affinity purification: Cells were centrifuged at 13000 rpm for 20 min and the supernatant was collected and purified using pre-packed column Hitrap Mabselect Sure. The procedure was as follows: before purification, the column was equilibrated with 5 column volumes of equilibration buffer (0.2 M Tris, 0.15 M NaCl, pH 7.2); the collected supernatant was passed through the column, and the column was washed with 10 column volumes of equilibration buffer to remove non-specifically bound proteins; the column was then washed with 5 column volumes of elution buffer (0.1 M sodium citrate, pH 3.5) and the eluate was collected; 80 μΐ of Tris (2 M Tris) was added per 1 ml of eluate, the eluate was concentrated using an ultrafiltration concentrator, and the concentration and purity were determined.

[0320] Ion exchange purification: the heterodimeric molecules in the bispecific molecules were separated from the homodimeric impurities by ion exchange chromatography.

[0321] Example 3. Affinity determination of IL-12 immunoconjugates to the receptor

[0322] The equilibrium dissociation constant (KD) of the antibodies of the application binding to human PD-1 or IL-12 receptor was determined using the Bio-Layer Interferometry assay. The affinity determination was performed according to the existing method (Estep, P, et al. High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013. 5(2): p. 270-8).

[0323] Affinity determination method of immunoconjugates to human PD-1 : the AHQ (Pall, 1502051) sensor was equilibrated in assay buffer for 30 minutes off-line, then on-line for 60 seconds to establish a baseline, and the purified antibody obtained as described above was loaded on the AHQ sensor (ForteBio) for the ForteBio affinity measurement. The sensor with the loaded immunoconjugate was then exposed to the antigen (including human PD-1 (ACRO, PD1-H5221), after which the sensor was transferred to assay buffer for dissociation rate measurement. The KD values were analyzed using the ForteBio analysis software.

[0324] Method for affinity determination of immunoconjugate to human IL-12 receptor: human biotinylated IL-12Rβ1 (ACRO, ILB-H52E1) or IL-12Rβ2 (ACRO, ILB-H52H6) was loaded to SA sensor (PALL, 18-5019), 100 nM immunoconjugate was placed in solution, and the experimental method was the same as above.

[0325] The results of detecting the affinity of antibodies are shown in Table 3:

[0326] Table 3. ForteBio detection of the affinity (equilibrium dissociation constant KD) of antigen-antibody binding Weak binding: refers to lower than the detection lower limit.

[0327] Example 4. In vitro activity determination of immunoconjugate

[0328] I. Detection of PD-1 activity using PD-L1αAPC / CHO-K1 cells and PD-1 effector Jurkat cells

[0329] PD-L1αAPC / CHO-K1 cells (Promega, J1250) are CHO-K1 cells stably expressing human PD-L1 and a cell surface protein that can activate cognate TCR independent of antigen. PD-1 effector Jurkat cells (Promega, J1250) are Jurkat T cells stably expressing human PD-1, TCR and NFAT-mediated luciferase.

[0330] When the above two cells are co-incubated, the PD-L1 on the surface of PD-L1αAPC / CHO-K1 cells binds to the PD-1 on the surface of PD-1 effector Jurkat cells, thereby inhibiting the TCR signaling in PD-1 effector Jurkat cells, and further inhibiting the expression of NFAT-mediated luciferase.

[0331] The antibody end of the αPD-1 / IL-12m immunoconjugate molecule that binds PD-1 can block the binding of PD-L1 to PD-1 when the above two cells are co-incubated, thereby activating TCR signaling and NFAT-mediated luciferase expression. The biological activity of the PD-1 antibody can be quantified by the luciferase reporter gene signal.

[0332] Experimental method:

[0333] 1. Configure Recovery Medium: 90% Ham's F-12 (Gibco, 11765-054) + 10% FBS (Gibco, 10091-148).

[0334] 2. Configure Assay Medium: 99% RPMI 1640 (Gibco, 22400-071) + 1% FBS (Gibco, 10091-148).

[0335] 3. Adjust PD-L1 alpha APC / CHO-K1 cell density with Recovery Medium to 40,000 cells per well in a volume of 100 μΐ in a 96-well flat bottom cell culture plate (Corning, 167008).

[0336] 4. Plate edge wells with an equal volume of Recovery Medium and incubate at 37°C, 5% C02 for 15-18 h.

[0337] 5. Remove 96-well flat bottom cell culture plate and discard media in wells, wash once with Assay Medium.

[0338] 6. Add 50 μΐ volume of diluted test immunoconjugate molecule to cell plate.

[0339] 7. Wash PD-1 effector Jurkat cells once with Assay Medium and resuspend cells in Assay Medium and adjust cell density to 50,000 cells per well in a volume of 50 μΐ to cell plate.

[0340] 8. Incubate cell plate at 37°C, 5% C02 for 6 h.

[0341] 9. Remove cell plate and equilibrate at room temperature for 15-20 min, add 100 μΐ Bio-Glo Luciferase Assay System reagent (Promega, G7940) per well, incubate at room temperature for 5 min in the dark. Remove 100 μΐ volume of solution from each well and add to a new 96-well white cell culture plate (Thermo, 136101). Read using a microplate reader (Molecular Devices) (settings: integration time 500 ms, read height 5.68 mm).

[0342] The results are shown in Figure 2. Figure 2 shows that the EC50 of the biological activity of the PD-1 antibody end of the αPD-1 / IL-12m immunoconjugate (1152, 1167) of the present study (i.e. the blocking effect of the PD-1 / PD-L1 signaling pathway) is equivalent to that of the parent anti-PD-1 monoclonal antibody IBI308 (Sintilimab), i.e. the biological activity of the two in blocking the PD-1 / PD-L1 signaling pathway is equivalent.

[0343] The results show that the PD-1 antibody end of the immunoconjugate molecule of the present application can effectively block the PD-1 / PD-L1 signaling pathway.

[0344] II. Detection of pSTAT4 signal in hPBMC cells by αPD-1 / IL-12m immunoconjugate molecules

[0345] IL-12 binds to the IL-12 receptor on the surface of T cells, which activates the JAK-STAT signaling pathway of T lymphocytes, and the level of STAT4 phosphorylation is an important indicator for judging the activation level of the signaling pathway.

[0346] Experimental method:

[0347] 1. hPBMC cell recovery

[0348] (1) Take liquid nitrogen frozen hPBMC cells (Miaoshun Biotechnology, PB100C-W) and melt quickly at 37°C.

[0349] (2) Slowly add the cells to 25 ml of CTS medium (Gibco, 0870112DK) preheated at 37°C.

[0350] (3) Centrifuge at 300g / 7min, remove the supernatant.

[0351] (3) Put 25 ml of CTS medium preheated at 37°C in a T175 culture flask (NUNC, 159910).

[0352] (4) Resuspend the hPBMC cells with 1 ml of CTS and transfer to the above T175 culture flask, and incubate overnight at 37°C in a 5% CO2 incubator.

[0353] 2. pSTAT4 experiment (all the following experimental steps are carried out in the dark)

[0354] (1) Resuspend the hPBMC cells with 1 ml of CTS and transfer to the above T175 culture flask, and incubate overnight at 37°C in a 5% CO2 incubator. TM488Antibody Labeling Kit (Invitrogen, A2018) to label PD-1 monoclonal antibody mAb07 (Innovent, sequence derived patent number US20160319019, SEQ ID: 85 & 93) to prepare Anti-PD-1-AF488 fluorescent antibody.

[0355] (2) Configure Test Medium: Add Anti-CD4-BV785 (BioLegend, 300554), Anti-CD8-BV421 (BioLegend, 301036), Anti-PD-1-AF488 (Innovent self-made) to CTS medium at 3:200; add Live / Dead yellow dye (Invitrogen, L34968) at 1:200.

[0356] (3) Configure FACS buffer: 98% PBS (Gibco, 10010-023) + 2% FBS (Gibco, 10091-148).

[0357] (4) Take out the T175 culture flask, take the suspended hPBMC cells, resuspend with Test Medium, adjust the concentration to 2x10 7 cells / ml.

[0358] (5) Use the resuspended hPBMC cell suspension to plate 96-well round-bottom cell culture plates (Costar, 3799) at a volume of 50 μl per well.

[0359] (6) Add 25 μl of different dilution concentrations of the immune conjugate to be tested to the 96-well plate, and incubate the sample to be tested with the cells at 37°C, 5% CO2 for 30 min.

[0360] (7) Centrifuge at 300g / 7min, remove the supernatant. Wash the cells with FACS buffer 2 times.

[0361] (8) Resuspend the cells with 4% tissue cell fixative (Solarbio, P1110) pre-cooled on ice at 180 μl / well for fixation, and incubate at 4°C for 15 min.

[0362] (9) Centrifuge at 500g / 4min, remove the supernatant.

[0363] (10) Add membrane-breaking solution (BD, 558050) at 180 μl / well, and incubate at 4°C in the dark for 1 h.

[0364] (11) Centrifuge at 500g / 4min, remove the supernatant.

[0365] (12) Configure Perm / wash buffer: Dilute Perm / wash buffer 10x (BD, 51-2091KZ) 10 times in ddH2O.

[0366] (13) Add Perm / wash Buffer at 200 μl / well, wash cells twice.

[0367] (14) Configure antibody staining solution: Add Anti-pSTAT4-AF647 (BD, 558137) at 1:100 in Perm / wash buffer.

[0368] (15) Add antibody staining solution at 50 μl / well, incubate at room temperature for 1 h in the dark.

[0369] (16) Wash cells twice with Perm / wash buffer.

[0370] (16) Resuspend cells with 100 μl Perm / wash buffer, and perform flow cytometry detection.

[0371] The results are shown in Figure 3. Figure 3 shows that the αPD-1 / IL-12m immunoconjugate molecules of the present study (1152, 1167, 1196) do not stimulate downstream pSTAT4 signaling of activated T lymphocytes in unpreactivated hPBMC. As a control, the wild-type IL-12-Fc molecule (62) activates downstream pSTAT4 signaling of activated T lymphocytes. Therefore, the αPD-1 / IL-12m immunoconjugate molecules of the present study do not cause downstream signaling activation of peripheral T cells in the peripheral environment in vivo, increasing the safety of the peripheral environment.

[0372] III. Ki67 signaling detection of αPD-1 / IL-12m immunoconjugate molecules in hPBMC cells

[0373] IL-12 binds to the IL-12 receptor on the surface of T cells, which activates the proliferation of T lymphocytes, and the expression level of Ki67 is an important indicator for judging the proliferation of T lymphocytes.

[0374] Experimental methods:

[0375] 1. hPBMC cell recovery

[0376] (1) Take liquid nitrogen frozen hPBMC cells (Miaoshun Biotechnology, PB100C-W), and quickly melt at 37°C.

[0377] (2) Slowly add the cells to 25 ml of CTS medium (Gibco, 0870112DK) preheated at 37°C.​

[0378] (3) Centrifuge 300g / 7min, remove supernatant.

[0379] (3) Put 25ml CTS medium pre-warmed at 37°C into T175 flask (NUNC, 159910).

[0380] (4) Resuspend hPBMC cells with 1ml CTS, transfer to T175 flask, incubate at 37°C 5% CO2 incubator overnight.

[0381] 2. Ki67 experiment

[0382] (1) Take out T175 flask, resuspend hPBMC cells with CTS medium, adjust concentration to 3x10 6 cells / ml.

[0383] (2) Plate 96-well round bottom cell culture plate (Costar, 3799) with resuspended hPBMC cell suspension, 100ul volume per well.

[0384] (3) Add 50ul volume of different dilution concentration of test immunconjugate to 96-well plate, incubate test sample with cells at 37°C 5% CO2 for 4 days.

[0385] (4) Label PD-1 monoclonal antibody mAb07 with Alexa Fluor TM 488 Antibody Labeling Kit (Invitrogen, A20181) to prepare Anti-PD-1-AF488 fluorescent antibody.

[0386] (5) Prepare FACS buffer: 98% PBS (Gibco, 10010-023) + 2% FBS (Gibco, 10091-148). Pre-cool FACS buffer in 4°C refrigerator.

[0387] (6) Prepare Test Medium: add Anti-CD4-BV421 (BioLegend, 300532) or Anti-CD4-AF700 (BioLegend, 300526), Anti-CD8-PE (Invitrogen, 12-0086-42), Anti-PD-1-AF488 (Innovent self-made) to FACS buffer at 1:100; add Live / Dead yellow dye (Invitrogen, L34968) at 1:500. Pre-cool Test Medium in 4°C refrigerator.

[0388] (7) Take out the cell culture plate, centrifuge at 300g / 7min, remove the supernatant. Wash the cells in the well with pre-cooled FACS buffer twice.

[0389] (8) Add Test Medium at 50μl / well, incubate in 4°C refrigerator for 1h.

[0390] (9) Centrifuge at 300g / 7min, remove the supernatant.

[0391] (10) Wash the cells in the well with pre-cooled FACS buffer twice.

[0392] (11) Prepare Fix / Perm Buffer: mix Fixation / Permeabilization Concentrate in eBioscience Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen, 00-5523-00) with eBioscience Fixation / Permeabilization Diluent at 1:3, and pre-cool in 4°C refrigerator. TM Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen, 00-5523-00) with eBioscience Fixation / Permeabilization Diluent at 1:3, and pre-cool in 4°C refrigerator.

[0393] (12) Prepare Perm / Wash Buffer: mix Permeabilization Buffer 10x in eBioscience Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen, 00-5523-00) with ddH2O at 1:9, and pre-cool in 4°C refrigerator. TM Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen, 00-5523-00) with eBioscience Fixation / Permeabilization Diluent at 1:3, and pre-cool in 4°C refrigerator.

[0394] (13) Add pre-cooled Fix / Perm Buffer at 180μl / well, incubate in 4°C refrigerator for 1h.

[0395] (14) Centrifuge at 500g / 4min, remove the supernatant.

[0396] (15) Wash the cells in the well with Perm / Wash Buffer twice.

[0397] (16) Prepare antibody staining solution: add Anti-Ki67-BV786 (BD, 563756) in Perm / Wash Buffer at 1:100.

[0398] (14) Add 50 μl / well of antibody staining solution, incubate at room temperature for 1 h in the dark, and wash twice with Perm / Wash Buffer.

[0399] (15) Resuspend the cells with 100 μl of Perm / Wash Buffer, and perform flow cytometry detection.

[0400] The results are shown in Figure 4. Figure 4 shows that the αPD-1 / IL-12m immunoconjugate of the present study had no obvious proliferative effect on CD4 + T and CD8 + T cells. Therefore, it will not cause the proliferation of peripheral T cells in the peripheral environment in vivo, and reduce systemic toxicity.

[0401] Four, detection of pSTAT4 signal of immunoconjugate molecules in activated hPBMC cells

[0402] After the activation of T lymphocytes, the effect of the immunoconjugate on the pSTAT4 signal of activated T lymphocytes was explored and verified under the action of PD-1.

[0403] 1. hPBMC cell recovery

[0404] (1) Take liquid nitrogen frozen hPBMC cells (Miaoshun Biotechnology, PB100C-W), and quickly melt at 37°C.

[0405] (2) Slowly add the cells to 25 ml of CTS medium (Gibco, 0870112DK) preheated at 37°C.

[0406] (3) Centrifuge at 300g / 7min, and remove the supernatant.

[0407] (3) Place 25 ml of CTS medium preheated at 37°C in a T175 culture flask (NUNC, 159910).

[0408] (4) Resuspend the hPBMC cells with 1 ml of CTS, and transfer to the above-mentioned T175 culture flask, and incubate at 37°C in a 5% CO2 incubator overnight.

[0409] 2. T lymphocyte activation and rest

[0410] (1) Take out the T175 culture flask, take the suspended hPBMC cells, resuspend in 10 ml of CTS medium, and count.

[0411] (2) Add an equal number of Dynabeads TM ​Human T-Activator CD3 / CD28 (Gibco, 11132D), activated at 37°C with 5% CO2 for 72 hours.

[0412] (3) Remove Dynabeads using a magnetic rack (STEMCELL, 18103).

[0413] (4) Wash the cells three times with CTS medium.

[0414] (3) Resuspend the activated cells in 25 ml of CTS medium and transfer them to a T175 culture flask. Incubate at 37°C and 5% CO2 for 48 h.

[0415] 3. pSTAT4 Experiment (All experimental steps below shall be performed in the dark)

[0416] (1) Using Alexa Fluor TM Anti-PD-1-AF488 fluorescent antibody was prepared by labeling PD-1 monoclonal antibody mAb07 with the 488Antibody Labeling Kit (Invitrogen, A20181).

[0417] (2) Prepare Test Medium: Add Anti-CD4-BV785 (BioLegend, 300554), Anti-CD8-BV421 (BioLegend, 301036), and Anti-PD-1-AF488 (Innovent self-made) to CTS medium at a ratio of 3:200; add Live / Dead Yellow dye (Invitrogen, L34968) at a ratio of 1:200.

[0418] (3) Remove the T175 culture flask, take the suspended hPBMC cells, resuspend them in Test Medium, and adjust the concentration to 2×10⁻⁶. 7 cells / ml.

[0419] (4) Spread 50 μl of resuspended hPBMC cell suspension onto a 96-well round-bottom cell culture plate (Costar, 3799) in each well.

[0420] (5) Add 25 μl of the different dilution concentrations of the immunoconjugate to be tested to a 96-well plate and incubate the sample and cells at 37°C for 30 min.

[0421] (6) Configure FACS buffer: 98% PBS (Gibco, 10010-023) + 2% FBS (Gibco, 10091-148).

[0422] (7) Centrifuge 300g / 7min, remove supernatant. Wash cells with FACS buffer twice.

[0423] (8) Resuspend and fix cells with 4% tissue cell fixative (Solarbio, P1110) pre-cooled on ice, 180ul / well, incubate at 4°C for 15min.

[0424] (8) Centrifuge 500g / 4min, remove supernatant.

[0425] (9) Add membrane disrupter (BD, 558050), 180ul / well, incubate at 4°C for 1h.

[0426] (10) Centrifuge 500g / 4min, remove supernatant.

[0427] (11) Prepare Perm / wash buffer: dilute Perm / wash buffer 10x (BD, 51-2091KZ) 10-fold in ddH2O.

[0428] (12) Add Perm / wash Buffer, 200ul / well, wash cells twice.

[0429] (13) Prepare antibody staining solution: add Anti-pSTAT4-AF647 (BD, 558137) in Perm / wash buffer at 1:100.

[0430] (14) Add antibody staining solution, 50ul / well, incubate at room temperature for 1h in the dark, wash with Perm / wash buffer twice.

[0431] (15) Resuspend cells with 100ul Perm / wash buffer, flow cytometry detection.

[0432] The results are shown in Figure 5. Figure 5 shows that the alphaPD-1 / IL-12m immunoconjugate of the present study has significantly stronger molecular activity in T cells (PD1 high CD4 + T and PD1 high CD8 + T) with high expression of PD-1 (PD1 low CD4 + T and PD1 low CD8 +T). As a control, the activity of wild type IL-12-Fc molecule (62) did not show significant change in T cells with high and low expression of PD-1. It is illustrated that the aPD-1 / IL-12m immunoconjugate of this study has significant selectivity for T cells with different expression of PD-1, thus having a larger safety window in vivo.

[0433] V. Ki67 signal detection of aPD-1 / IL-12m immunoconjugate molecules in activated hPBMC cells

[0434] IL-12 binds to IL-12 receptors on the surface of T cells, which activates the proliferation of T lymphocytes, and the expression level of Ki67 is an important indicator for evaluating the proliferation of T lymphocytes.

[0435] Experimental method:

[0436] 1. hPBMC cell recovery

[0437] (1) Take liquid nitrogen frozen hPBMC cells (Miaoshun Biotechnology, PB100C-W), and melt quickly at 37°C.

[0438] (2) Slowly add the cells to 25 ml of CTS medium (Gibco, 0870112DK) preheated at 37°C.

[0439] (3) Centrifuge at 300g / 7min, remove the supernatant.

[0440] (3) Put 25 ml of CTS medium preheated at 37°C in a T175 culture flask (NUNC, 159910).

[0441] (4) Resuspend the hPBMC cells with 1 ml of CTS, transfer to the above T175 culture flask, and incubate at 37°C in a 5% CO2 incubator overnight.

[0442] 2. T lymphocyte activation and rest

[0443] (1) Take out the T175 culture flask, take the suspended hPBMC cells, resuspend in 10 ml of CTS medium and count.

[0444] (2) Add Dynabeads TM Human T-Activator CD3 / CD28 (Gibco, 11132D) to the cell solution, and activate for 48 h.

[0445] (3) Remove the Dynabeads with a magnetic stand (STEMCELL, 18103).

[0446] (4) Wash the cells with CTS medium 3 times.

[0447] (3) Resuspend the activated cells with 25ml CTS medium and transfer to T175 culture flask, incubate at 37°C 5% CO2 for 72h.

[0448] 3. Ki67 experiment (all the following experimental steps are performed in the dark)

[0449] (1) Take out the T175 culture flask, resuspend with CTS medium, adjust the concentration to 3x10 6 cells / ml.

[0450] (2) Use the resuspended hPBMC cell suspension to plate 96-well round-bottom cell culture plates (Costar, 3799), 100ul volume per well.

[0451] (3) Add 50ul volume of different dilution concentrations of the test immune conjugate to the 96-well plate, respectively, and incubate the test sample with the cells at 37°C 5% CO2 for 4 days.

[0452] (4) Label PD-1 monoclonal antibody mAb07 with Alexa Fluor TM 488 Antibody Labeling Kit (Invitrogen, A20186) to prepare Anti-PD-1-AF488 fluorescent antibody.

[0453] (5) Prepare FACS buffer: 98% PBS (Gibco, 10010-023) + 2% FBS (Gibco, 10091-148). Pre-cool the FACS buffer in the 4°C refrigerator.

[0454] (6) Prepare Test Medium: Add Anti-CD4-BV421 (BioLegend, 300532), Anti-CD8-PE (Invitrogen, 12-0086-42), Anti-PD-1-AF488 (Innovent self-made) to the FACS buffer at 1:100; add Live / Dead yellow dye (Invitrogen, L34968) at 1:500. Pre-cool the Test Medium in the 4°C refrigerator.

[0455] (7) Take out the cell culture plate, centrifuge at 300g / 7min, remove the supernatant. Wash the cells in the wells with pre-cooled FACS buffer twice.

[0456] (8) Add Test Medium according to 50ul / well, incubate in the 4°C refrigerator in the dark for 1h.

[0457] (9) Centrifuge 300g / 7min, remove supernatant.

[0458] (10) Wash cells in wells twice with pre-chilled FACS buffer.

[0459] (11) Prepare Fix / Perm Buffer: Mix Fixation / Permeabilization Concentrate from eBioscience Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen, 00-5523-00) with Fixation / Permeabilization Diluent at 1:3 ratio, and pre-chill in 4°C refrigerator. TM

[0460] (12) Prepare Perm / Wash Buffer: Mix Permeabilization Buffer 10x from eBioscience Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen, 00-5523-00) with ddH2O at 1:9 ratio, and pre-chill in 4°C refrigerator. TM

[0461] (13) Add pre-chilled Fix / Perm Buffer at 180μl / well, and incubate at 4°C in dark for 1h.

[0462] (14) Centrifuge 500g / 4min, remove supernatant.

[0463] (15) Wash cells in wells twice with Perm / Wash Buffer.

[0464] (16) Prepare antibody staining solution: Add Anti-Ki67-BV786 (BD, 563756) at 1:100 in Perm / Wash Buffer.

[0465] (14) Add antibody staining solution at 50μl / well, and incubate at room temperature in dark for 1h.

[0466] (15) Wash cells twice with Perm / Wash Buffer.

[0467] (16) Resuspend cells with 100μl Perm / Wash Buffer, and perform flow cytometry detection.

[0468] ​​The results are shown in Figure 6. Figure 6 shows that the aPD-1 / IL-12m immunoconjugate of the present study has a certain proliferative effect on CD4 + T and CD8 + T cells. The proliferative effect is significantly reduced compared to the wild-type IL-12-Fc molecule (62), thus reducing the toxicity in vivo.

[0469] Six, detection of pSTAT4 signal in activated mouse spleen cells by mouse surrogate molecule 1 (1182) and surrogate molecule 2 (1183) of the immunoconjugate molecule

[0470] After the activation of T lymphocytes, the effect of the immunoconjugate on the pSTAT4 signal of activated T lymphocytes was explored and verified under the action of PD-1.

[0471] 1. Obtain spleen cells

[0472] (1) Prepare FACS buffer: 98% PBS (Gibco, 10010-023) + 2% FBS (Gibco, 10091-148).

[0473] (2) Prepare Growth Medium: 89% RPMI 1640 (Gibco, 22400-071) + 10% FBS + 1% Pen-Strep (Gibco, 15070-063).

[0474] (3) Under sterile conditions, remove the spleen of C57BL / 6N mice (Beijing VitoLihua, SPF level, female mice, certificate number 110011231105689135) and grind into single cells, dissolve in FACS buffer and pass through a 0.45 μm filter (Falcon, 352340).

[0475] (4) Centrifuge at 500g for 5min, remove the supernatant.

[0476] (5) Add ACK lysis buffer (Gibco, A10492-01) to the cells to lyse the red blood cells for 3 minutes, and add Growth Medium to terminate the reaction.

[0477] (6) Centrifuge at 500g for 5min, remove the supernatant, and wash the cells once with FACS buffer, and pass the cells through a 0.45 μm filter.

[0478] (7) Resuspend the cells in Growth Medium and adjust the cell concentration to 1.5x10 6 cells / ml.

[0479] 2. T lymphocyte activation and rest

[0480] (1) Add Anti-Mo CD3e (Invitrogen, 14-0031-86) and Anti-Mo CD28 (Invitrogen, 16-0281-86) to the spleen cell solution at a ratio of 1:1000 by volume, and add rmIL-2 (Sino Biological, 51061-MNAE) to a final concentration of 10 ng / ml.

[0481] (2) Add the above cell mixture solution to a 24-well plate (NEST, 702001) at 2 ml per well, and incubate at 37°C in a 5% CO2 incubator for 72 h.

[0482] (3) Collect the cell solution in the 24-well plate and wash the cells with Growth Medium three times.

[0483] (3) Resuspend the activated cells with Growth Medium to adjust the concentration to 5 x 10 6 cells / ml and transfer to a T75 culture flask (NUNC, 156499) for overnight incubation at 37°C in a 5% CO2 incubator.

[0484] 3. pSTAT4 experiment (all the following experimental steps are carried out in the dark)

[0485] (1) Prepare Test Medium: Add Anti-CD3-AF700 (BioLegend, 100216), Anti-CD4-BV421 (BioLegend, 100563), Anti-CD8-PE (Invitrogen, 12-0081-81), Anti-PD-1-FITC (BioLegend, 135214) to Growth Medium at a ratio of 3:200; add Live / Dead yellow dye (Invitrogen, L34968) at a ratio of 1:300.

[0486] (2) Take out the cells in the T75 culture flask and resuspend them with Test Medium, adjusting the concentration to 2 x 10 7 cells / ml.

[0487] (3) Use the resuspended cell suspension to plate a 96-well round-bottom cell culture plate (Costar, 3799) at a volume of 50 μl per well.

[0488] (4) Add 25 μl of different dilution concentrations of the test immunocompounds to the 96-well plate, respectively, and incubate the test samples with the cells at 37°C for 30 min.

[0489] (5) Centrifuge 400g / 4min, remove supernatant. Wash cells in plate twice with FACS buffer.

[0490] (6) Resuspend cells with 4% tissue cell fixative (Solarbio, P1110) pre-cooled on ice, fix cells according to 180ul / well, incubate at 4°C for 15min.

[0491] (7) Centrifuge 500g / 4min, remove supernatant.

[0492] (8) Add membrane break solution (BD, 558050) according to 180ul / well, incubate at 4°C for 1h in the dark.

[0493] (9) Centrifuge 500g / 4min, remove supernatant.

[0494] (10) Configure Perm / wash buffer: dilute Perm / wash buffer 10x (BD, 51-2091KZ) 10 times in ddH2O.

[0495] (11) Add Perm / wash Buffer according to 200ul / well, wash cells twice.

[0496] (12) Configure antibody staining solution: add Anti-pSTAT4-AF647 (BD, 558137) to Perm / wash buffer at 1:100.

[0497] (13) Add antibody staining solution according to 50ul / well, incubate at room temperature for 1h in the dark, wash twice with Perm / wash buffer.

[0498] (14) Resuspend cells with 100ul Perm / wash buffer, flow cytometry detection.

[0499] The results are shown in Figure 7. Figure 7 shows that the mouse surrogate molecules 1 (1182) and 2 (1183) of the αPD-1 / IL-12m immunoconjugate of the present study have significantly stronger molecular activity in T cells (PD1 high CD4 + T and PD1 high CD8 + T) with high expression of PD-1 than in T cells (PD1 low CD4 + T and PD1 low CD8 +T). As a control, the activity of wild-type mIL-12-Fc molecule (98) did not show significant change in T cells with high and low expression of PD-1. It is illustrated that the aPD-1 / IL-12m immunoconjugate of the present study has obvious selectivity for T cells with different expression amounts of PD-1, thus having a larger safety window in vivo.

[0500] Seven, detection of pSTAT4 signal of mouse surrogate molecule 3 (1193) and surrogate molecule 4 (1194) of immunoconjugate molecules in activated mouse spleen cells

[0501] After the T lymphocytes are activated, the effect of the immunoconjugate on the pSTAT4 signal of the activated T lymphocytes is explored and verified under the action of PD-1.

[0502] 1. Obtain spleen cells

[0503] (1) Prepare FACS buffer: 98% PBS (Gibco, 10010-023) + 2% FBS (Gibco, 10091-148).

[0504] (2) Prepare Growth Medium: 89% RPMI 1640 (Gibco, 22400-071) + 10% FBS + 1% Pen-Strep (Gibco, 15070-063).

[0505] (3) Under a sterile environment, take out the spleen of the Balb / c mouse (Shanghai South Model Organism, SPF level, female mouse, certificate number 20190002026327) into which hPD-1 is introduced, grind into single cells, dissolve in FACS buffer and pass through a 0.45 μm screen (Falcon, 352340).

[0506] (4) Centrifuge at 500g / 5min, remove the supernatant.

[0507] (5) Add ACK lysis buffer (Gibco, A10492-01) to the cells to lyse the red blood cells for 3 min, and add Growth Medium to terminate the reaction.

[0508] (6) Centrifuge at 500g / 5min, remove the supernatant, and wash the cells once with FACS buffer, and pass the cells through a 0.45 μm screen.

[0509] (7) Resuspend the cells in Growth Medium and adjust the cell concentration to 1.5×10 6 cells / ml.

[0510] 2. T lymphocyte activation and rest

[0511] (1) Add Anti-Mo CD3e (Invitrogen, 14-0031-86) and Anti-Mo CD28 (Invitrogen, 16-0281-86) to the spleen cell solution at a ratio of 1:1000 by volume, and add rmIL-2 (Sino Biological, 51061-MNAE) to a final concentration of 10 ng / ml.

[0512] (2) Add the above cell mixture solution to a 24-well plate (NEST, 702001) at 2 ml per well, and incubate in a 37°C 5% CO2 incubator for 48 h.

[0513] (3) Collect the cell solution in the 24-well plate and wash the cells with Growth Medium 3 times.

[0514] (4) Resuspend the activated cells with Growth Medium to adjust the concentration to 5×10 6 cells / ml and transfer to a T75 culture flask (NUNC, 156499), and incubate at 37°C 5% CO2 overnight.

[0515] 3. pSTAT4 experiment (all the following experimental steps are carried out in the dark)

[0516] (1) Label the PD-1 monoclonal antibody mAb07 (Innovent self-made) with an Alexa Fluor TM 488 Antibody Labeling Kit (Thermo Fisher, A20181) to prepare Anti-PD-1-AF488 fluorescent antibody.

[0517] (2) Prepare Test Medium: add Anti-CD3-AF700 (BioLegend, 100216), Anti-CD4-BV421 (BioLegend, 100563), Anti-CD8-PE (Invitrogen, 12-0081-81), Anti-PD-1-AF488 (Innovent self-made) to Growth Medium at a ratio of 3:200; add Live / Dead yellow dye (Invitrogen, L34968) at a ratio of 1:300.

[0518] (3) Take out the cells in the T75 culture flask, resuspend with Test Medium, and adjust the concentration to 2×10 7 cells / ml.

[0519] (4) Resuspend the cells with 50 μl volume per well in 96-well round bottom cell culture plates (Costar, 3799).

[0520] (5) Add 25 μl volume of different dilution concentration of the test immunoconjugate to the 96-well plate respectively, and incubate the test sample with the cells at 37°C for 30 min in 5% CO2.

[0521] (6) Centrifuge at 400g for 4 min, remove the supernatant, and wash the cells with FACS buffer twice.

[0522] (7) Resuspend the cells with 180 μl / well of 4% tissue cell fixation solution (Solarbio, P1110) pre-cooled on ice for fixation, and incubate at 4°C for 15 min.

[0523] (8) Centrifuge at 500g for 4 min, remove the supernatant.

[0524] (9) Add 180 μl / well of membrane-breaking solution (BD, 558050), and incubate at 4°C for 1 h in the dark.

[0525] (10) Centrifuge at 500g for 4 min, remove the supernatant.

[0526] (11) Prepare Perm / wash buffer: dilute Perm / wash buffer 10x (BD, 51-2091KZ) 10-fold in ddH2O.

[0527] (12) Wash the cells twice with 200 μl / well of Perm / wash Buffer.

[0528] (13) Prepare antibody staining solution: add Anti-pSTAT4-AF647 (BD, 558137) at 1:100 in Perm / wash buffer.

[0529] (14) Add 50 μl / well of the antibody staining solution, and incubate at room temperature in the dark for 1 h, and wash twice with Perm / wash buffer.

[0530] (15) Resuspend the cells with 100 μl of Perm / wash buffer, and perform flow detection.

[0531] The results are shown in FIG. 8. FIG. 8 shows that the mouse surrogate molecules 3 (1193) and 4 (1194) of the αPD-1 / IL-12m immunoconjugate of the present study have a higher binding activity to T cells (PD1 high CD4 + T and PD1 high CD8 +In T), the molecular activity was significantly stronger in T cells with low expression of PD-1 (PD1 low CD4 + T and PD1 low CD8 + T). As a control, the activity of the wild-type mIL-12-Fc molecule (98) did not change significantly in T cells with high and low expression of PD-1. This indicates that the aPD-1 / IL-12m immunoconjugate of the present study has obvious selectivity for T cells with different expression amounts of PD-1, and thus has a larger safety window in vivo. At the same time, the activity and selectivity of 1193 and 1194 for T cells with different expression amounts of PD-1 in Figure 7 are very similar to the aPD-1 / IL-12m immunoconjugate in Figure 4, indicating that 1193 and 1194 can replace the human-derived molecule to verify the efficacy and safety in vivo.

[0532] Example 5. In vivo efficacy experiment of immunoconjugate

[0533] I. Efficacy and safety of mouse surrogate molecule 4 (1194) of immunoconjugate molecule in CT26 model

[0534] To demonstrate the efficacy of aPD-1 / IL-12m immunoconjugate in vivo, the anti-tumor efficacy of the aPD-1 / IL-12m immunoconjugate in the present study was determined by inoculating hPD-1 knock-in Balb / c mice (Shanghai South Model, SPF level, female, certificate number 2019002027654) with CT26 cells (mouse intestinal cancer cell line, ATCC). Due to species differences, mouse surrogate molecule 4 (1194) was used instead of the human-derived conjugate to verify the efficacy and safety in this experiment.

[0535] CT26 cells were routinely passaged for subsequent in vivo experiments. The cells were collected by centrifugation, resuspended in 20% matrigel (CORNING, 356231) / PBS (1x) (Gibco, 10010-023) at a concentration of 5x10 6 On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of hPD-1 knock-in Balb / c mice to establish a CT26 tumor-bearing mouse model.

[0536] The tumor volume of each mouse was detected 6 days after tumor cell inoculation, and the mice were grouped (7 mice per group). The dosages and modes of administration are shown in Table 4.

[0537] Table 4. Grouping, dosages, and modes of administration in CT26 in vivo experiment 1h-IgG is isotype control antibody, Equitech-Bio, SLH56, 211110-0256.

[0538] The three groups in Table 4 were dosed at day 6 and day 13 after CT26 cell inoculation, and the tumor volume and body weight of mice were monitored twice a week, as shown in Figure 9, and the monitoring was ended after 23 days.

[0539] The relative tumor inhibition rate (TGI%) was calculated at day 13 after inoculation, and the calculation formula was as follows: TGI% = 100% x (tumor volume of control group - tumor volume of treatment group) / (tumor volume of control group - tumor volume before drug administration of control group). Tumor volume determination: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured by vernier caliper, and the tumor volume was calculated according to the following formula: V = L x W 2 / 2. The body weight was measured by electronic balance. When the tumor volume of a mouse exceeded 2000 mm 3 The mouse will be euthanized. If more than half of the mice in the group die, the tumor growth curve of the whole group will not be displayed at this time point.

[0540] The tumor inhibition rate results are shown in Table 5 and Figure 9A: at day 13 after inoculation, compared with the h-IgG group, the tumor inhibition rates of the 1194-10 mg / kg and 1194-30 mg / kg groups were 82.49% and 87.85%, respectively. The results of the detection of the body weight of the mice (Figure 9B) showed that there was no significant difference in the body weight of the mice at day 23 after inoculation.

[0541] Table 5, Anti-tumor efficacy statistics (day 13 after inoculation) 1 h-IgG is isotype control antibody, Equitech-Bio, SLH56, 211110-0256.

[0542] II. Efficacy and safety of mouse surrogate molecules 3 (1193) and 4 (1194) of immunoconjugate molecules in EMT6 model

[0543] In order to further prove the efficacy of the αPD-1 / IL-12m immunoconjugate in vivo, the anti-tumor efficacy of the αPD-1 / IL-12m immunoconjugate in this study was determined by inoculating EMT6 cells (mouse breast cancer cell line, ATCC) with hPD-1 knock-in Balb / c mice (Shanghai South Model, SPF level, female mice, certificate number 2019002026327) to determine the anti-tumor efficacy of the αPD-1 / IL-12m immunoconjugate in this study. Due to the species difference, mouse surrogate molecule 3 (1193) and surrogate molecule 4 (1194) were used instead of human-derived conjugates for efficacy and safety verification in this experiment.

[0544] EMT6 cells were routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation, resuspended in PBS (1x) (Gibco, 10010-023) to prepare a cell suspension with a concentration of 5x10 6 On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right flank of the hPD-1 knock-in Balb / c mice to establish the EMT6 tumor-bearing mouse model.

[0545] The tumor volume of each mouse was measured 7 days after tumor cell inoculation, and the mice were grouped (7 mice per group). The dosing amount and method are shown in Table 6.

[0546] Table 6: Grouping, dosing amount, and method of EMT6 in vivo experiment 1 h-IgG is an isotype control antibody, manufacturer Equitech-Bio, item number SLH56, batch number 211110-0256.

[0547] The four groups in Table 6 were dosed on days 7, 11, 14, and 18 after EMT6 cell inoculation, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figure 10, until the end of the experiment on day 32.

[0548] The relative tumor inhibition rate (TGI%) was calculated on day 14 after inoculation, and the calculation formula is as follows: TGI% = 100% x (tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume before dosing of the control group). Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula: V = L x W 2 / 2. The body weight was measured using an electronic balance. If the tumor volume exceeds 2000 mm 3 The mouse will be euthanized. If more than half of the group dies, the tumor growth curve of the entire group will not be shown at that time point.

[0549] The tumor inhibition rate results are shown in Table 7 and Figure 10A: on day 14 after inoculation, compared with the h-IgG group, the tumor inhibition rates of the 1193-10 mg / kg, 1194-3 mg / kg, and 1194-10 mg / kg groups were 87.44%, 65.82%, and 99.20%, respectively. The results of the body weight detection of the mice (Figure 10B) showed that on day 32 after inoculation, there was no significant difference in the body weight of the mice.

[0550] Table 7: Anti-tumor efficacy statistics (on day 14 after inoculation) 1 h-IgG is an isotype control antibody, manufacturer Equitech-Bio, item number SLH56, batch number 211110-0256.

[0551] III. Pharmacodynamics and safety of mouse surrogate molecule 4 (1194) of immunoconjugate molecule in LLC1 model

[0552] To further demonstrate the pharmacodynamics of the αPD-1 / IL-12m immunoconjugate in vivo, the anti-tumor pharmacodynamics of the αPD-1 / IL-12m immunoconjugate in this study was determined by inoculating LLC1 cells (mouse lung cancer cell line, ATCC, item number: CRL-1642, batch number: 63332958) with a large number of immunosuppressive MDSCs in the tumor microenvironment into hPD-1 knock-in C57BL / 6N mice (Baiosaituo, SPF level, female, certificate number 20190002029374). Due to the species difference, mouse surrogate molecule 4 (1194) was used instead of the human-derived conjugate for pharmacodynamics and safety verification in this experiment.

[0553] LLC1 cells were routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation, resuspended in PBS (1×) (Gibco, 10010-023), and prepared into a cell suspension with a cell concentration of 0.8×10 6 On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of the hPD-1 knock-in C57BL / 6N mice to establish the LLC1 tumor-bearing mouse model.

[0554] The tumor volume of each mouse was detected 8 days after tumor cell inoculation, and the mice were grouped (7 mice per group). The dosing amount and method are shown in Table 8.

[0555] Table 8. Grouping, dosing amount, and method of LLC1 in vivo experiment 1 : h-IgG is the isotype control antibody, manufacturer Equitech-Bio, item number SLH56, batch number 211110-0256.

[0556] The two groups in Table 8 were dosed on days 8, 12, 15, and 19 after LLC1 cell inoculation, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figure 11, and the monitoring was stopped after 25 days.

[0557] The relative tumor inhibition rate (TGI%) was calculated on day 25 after inoculation, and the calculation formula was as follows: TGI% = 100% × (tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume before dosing of the control group). Tumor volume determination: the maximum long axis (L) and the maximum wide axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula: V = L × W 2 / 2. The body weight was measured using an electronic balance. When the tumor volume exceeded 2000 mm 3The mice will be euthanized. If more than half of the group dies, the tumor growth curve of the whole group does not show at this time point.

[0558] The tumor inhibition rate results are shown in Table 9 and Figure 11A: on day 25 after inoculation, the tumor inhibition rate of the 1194 group was 52.06% compared with the h-IgG group. The results of detecting the weight of mice (Figure 11B) showed that on day 25 after inoculation, there was no significant difference in the weight of mice.

[0559] Table 9, Anti-tumor drug efficacy statistics (day 25 after inoculation) 1 : h-IgG is the isotype control antibody, manufacturer Equitech-Bio, item number SLH56, batch number 211110-0256.

[0560] Four, efficacy and safety of immunoconjugate molecules (1167) in A375 model

[0561] In order to further prove that the in vivo efficacy of the αPD-1 / IL-12m immunoconjugate is better than that of the parent anti-PD-1 monoclonal antibody (Sintilimab, also known as IBI308), A375 cells (human melanoma cell line, ATCC, item number: CRL-1619TM, batch number: 61573377) were used to inoculate NOG mice (Beijing Vivotec Biotech, SPF level, female mice, certificate number 110011231109812174) to determine the anti-tumor efficacy of the αPD-1 / IL-12m immunoconjugate (1167) in this study.

[0562] Recovery hPBMC (AllCells, FPB004F-C) was resuspended in PBS (1x) (Gibco, 10010-023) to prepare a cell suspension with a cell concentration of 20x10 6 On day -3, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of the NOG mouse to establish a humanized NOG mouse model.

[0563] A375 cells were routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation, and the A375 cells were resuspended in 50% matrigel (CORNING, 356231) / 50% PBS (1x) (Gibco, 10010-023) to prepare a cell suspension with a cell concentration of 30x10 6 On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of the NOG mouse to establish a humanized A375 tumor-bearing mouse model.

[0564] The tumor volume and the proportion of hPBMC in blood of each mouse were detected 7 days after tumor cell inoculation, and the mice were grouped (7 mice per group). The dosages and administration methods are shown in Table 10.

[0565] Table 10. Grouping, dosages and administration methods of A375 in vivo experiment 1 : h-IgG is an isotype control antibody, manufacturer Equitech-Bio, product number SLH56, batch number 211110-0256. 2 : IBI308 is Sintilimab, which is a marketed PD-1 monoclonal antibody drug.

[0566] The four groups in Table 10 were administered on the 7th and 14th day after A375 cell inoculation, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figure 12, and the monitoring was stopped after 28 days.

[0567] The relative tumor inhibition rate (TGI%) was calculated on the 14th day after inoculation, and the calculation formula was as follows: TGI% = 100% x (tumor volume of control group - tumor volume of treatment group) / (tumor volume of control group - tumor volume before administration of control group). Tumor volume determination: the maximum long axis (L) and the maximum wide axis (W) of the tumor were measured by vernier caliper, and the tumor volume was calculated according to the following formula: V = L x W 2 / 2. The body weight was measured by electronic balance. If the tumor volume of a mouse exceeded 2000 mm 3 The mouse would be euthanized. If more than half of the mice in a group died, the tumor growth curve of the entire group would not be displayed at that time point.

[0568] The tumor inhibition rate results are shown in Table 11 and Figure 12A: on the 14th day after inoculation, compared with the h-IgG group, the tumor inhibition rates of the IBI308, 1167-3 mg / kg and 1167-10 mg / kg groups were 19.16%, 47.08% and 85.26%, respectively. The results of the detection of the body weight of the mice (Figure 12B) showed that on the 28th day after inoculation, there was no significant difference in the body weight of the mice.

[0569] Table 11. Anti-tumor efficacy statistics (14 days after inoculation) 1 : h-IgG is an isotype control antibody, manufacturer Equitech-Bio, product number SLH56, batch number 211110-0256.

[0570] V. Efficacy and safety of immunoconjugate molecules (1167) in BxPC-3 model

[0571] To further demonstrate the efficacy of the aPD-1 / IL-12m immunoconjugate in vivo, the anti-tumor efficacy of the aPD-1 / IL-12m immunoconjugate (1167) in this study was determined using BxPC-3 cells (human pancreatic cancer cell line, ATCC) to inoculate NOG mice (Beijing Vivotecno, SPF level, female mice, license number 110011231109964254).

[0572] Resuscitate hPBMC (AllCells, FPB004F-C) and resuspend in PBS (1x) (Gibco, 10010-023) to prepare a cell suspension with a cell concentration of 20 x 106 6 On day -3, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of the NOG mice to establish a humanized NOG mouse model.

[0573] BxPC-3 cells were routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation, and the BxPC-3 cells were resuspended in 50% matrigel (CORNING, catalog number 356231) / 50% PBS (1x) (Gibco, 10010-023) to prepare a cell suspension with a cell concentration of 20 x 106 6 On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of the NOG mice to establish a BxPC-3 tumor-bearing humanized mouse model.

[0574] After 8 days of tumor cell inoculation, the tumor volume of each mouse and the proportion of hPBMC in the blood were detected, and the mice were grouped (7 mice per group). The dosages and modes of administration are shown in Table 12.

[0575] Table 12, grouping, dosages, and modes of administration for BxPC-3 in vivo experiments 1 h-IgG is the isotype control antibody, manufacturer Equitech-Bio, catalog number SLH56, batch number 211110-0256.

[0576] The three groups in Table 12 were administered on day 8 after BxPC-3 cell inoculation, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figure 13, and the monitoring was stopped after 25 days.

[0577] The relative tumor inhibition rate (TGI%) was calculated on day 21 after inoculation, and the calculation formula was as follows: TGI% = 100% x (tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume before administration of the control group). Tumor volume determination: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula: V = L*W 2 / 2. Body weight was measured by using an electronic balance. Tumor size was measured by using a digital caliper. Tumor volume was calculated by using the formula: V = 0.5ab2, where a is the long diameter and b is the short diameter of the tumor. 3 The mice were euthanized if more than half of the mice in a group died. The tumor growth curve of the whole group was not displayed at this time point.

[0578] The tumor inhibition rate results are shown in Table 13 and Figure 13A: on day 21 after inoculation, the tumor inhibition rates of the 1167-3 mg / kg and 1167-10 mg / kg groups were 85.00% and 98.63%, respectively, compared with the h-IgG-10 mg / kg group. The results of the detection of the body weight of the mice (Figure 13B) showed that on day 25 after inoculation, there was no significant difference in the body weight of the mice.

[0579] Table 13, Anti-tumor efficacy statistics (on day 21 after inoculation) 1 : h-IgG is the isotype control antibody, manufacturer Equitech-Bio, product number SLH56, batch number 211110-0256.

[0580] Six, efficacy and safety of mouse surrogate molecule 4 (1194) of immunoconjugate molecules in the MC38 model

[0581] In order to prove the efficacy of the aPD-1 / IL-12m immunoconjugate in vivo and the safety of high-dose, the anti-tumor efficacy and drug tolerance of the aPD-1 / IL-12m immunoconjugate in this study were determined by inoculating MC38 cells (mouse intestinal cancer cell line, ATCC) into hPD-1 knock-in C57BL / 6J mice (Shanghai South Model, SPF level, female mice, certificate number 20190002032685). Due to the species difference, mouse surrogate molecule 4 (1194) was used instead of human conjugate to verify the efficacy and safety in this experiment.

[0582] The MC38 cells were routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation, resuspended in PBS (1x) (Gibco, 10010-023), and prepared into a cell suspension with a cell concentration of 4x10 6 On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of the hPD-1 knock-in C57BL / 6J mice to establish the MC38 tumor-bearing mouse model.

[0583] The tumor volume of each mouse was detected 7 days after tumor cell inoculation, and the mice were grouped (6 mice per group). The dosing amount and method are shown in Table 14.

[0584] Table 14, grouping, dosing amount and method of MC38 in vivo experiment 1h-IgG is isotype control antibody, vendor Equitech-Bio, Cat# SLH56, Lot# 211110-0256.

[0585] The four groups in Table 14 were dosed on days 7, 14 and 21 after MC38 cell inoculation, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figure 14, and the monitoring was ended after 28 days.

[0586] The relative tumor inhibition rate (TGI%) was calculated on day 28 after inoculation, and the calculation formula was as follows: TGI% = 100% x (tumor volume of control group - tumor volume of treatment group) / (tumor volume of control group - tumor volume before administration of control group). Tumor volume determination: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured by vernier caliper, and the tumor volume was calculated according to the following formula: V = L x W 2 / 2. The body weight was measured by electronic balance. If the tumor volume of a mouse exceeded 2000 mm 3 The mouse will be euthanized. If more than half of the mice in the group die, the tumor growth curve of the entire group will not be displayed at that time point.

[0587] The tumor inhibition rate results are shown in Table 15 and Figure 14A: on day 28 after inoculation, compared with the h-IgG group, the tumor inhibition rates of the 1194-10 mg / kg, 1194-30 mg / kg and 1194-90 mg / kg groups were 90.24%, 101.80% and 103.60%, respectively. The results of the detection of the body weight of the mice (Figure 14B) showed that on day 28 after inoculation, there was no significant difference in the body weight of the mice.

[0588] Table 15, Anti-tumor drug efficacy statistics (on day 28 after inoculation) 1 h-IgG is isotype control antibody, vendor Equitech-Bio, Cat# SLH56, Lot# 211110-0256.

[0589] Sequence Listing

Claims

1. An IL-12 mutein comprising an IL-12 p40 subunit variant and / or an IL-12 p35 subunit variant, wherein the (i) IL-12 p40 subunit variant comprises a K84E substitution and a substitution of the sequence segment “(SKREKKD)” at positions 259-265 to “(DNTEG)”; or the IL-12 p40 subunit variant comprises a K84E substitution and a deletion of the amino acids at positions 15-18; optionally the IL-12 p40 subunit variant further comprises a C252S substitution; (ii) IL-12 p35 subunit variant comprises a K170A substitution; wherein the amino acid positions of the IL-12 p40 subunit variant are determined with reference to the amino acid sequence set forth in SEQ ID NO: 4, and the amino acid positions of the IL-12 p35 subunit variant are determined with reference to the amino acid sequence set forth in SEQ ID NO:

3.

2. The IL-12 mutein of claim 1, wherein the IL-12 p40 subunit variant and the IL-12 p35 subunit variant are linked by a linker, for example the C-terminus of the IL-12 p40 subunit variant is linked to the N-terminus of the IL-12 p35 subunit variant by a linker; optionally, wherein the linker comprises (GGGGS) n wherein n = an integer from 1-10, for example 1, 2, 3, 4, 5, or 6; more preferably, the linker is GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 42).

3. The IL-12 mutein of claim 1 or 2, wherein the IL-12 p40 subunit variant comprises the amino acid sequence set forth in SEQ ID NO: 24, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or the IL-12 p40 subunit variant comprises the amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; and / or the IL-12 p35 subunit variant comprises the amino acid sequence set forth in SEQ ID NO: 23, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; optionally wherein the IL-12 p40 subunit variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11 or 24; and the IL-12 p35 subunit variant comprises or consists of the amino acid sequence set forth in SEQ ID NO:

23.

4. The IL-12 mutein of any one of claims 1-3, wherein the IL-12 mutein comprises (i) at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 41, and comprises a K84E substitution in the IL-12 p40 subunit variant and a substitution of the sequence segment “(SKREKKD)” at positions 259-265 to “(DNTEG)” in the IL-12 p40 subunit variant, and a K170A substitution in the IL-12 p35 subunit variant; or (ii) has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 40, and comprises a K84E substitution in the IL-12p40 subunit variant and a K170A substitution in the IL-12p35 subunit variant; Optionally, the IL-12 mutein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 40 or 41.

5. An immunoconjugate comprising the IL-12 mutein of any one of claims 1-4, and an antigen binding region that specifically binds PD-1; Optionally, wherein the antigen binding region that specifically binds PD-1 comprises 3 heavy chain variable region- contained complementarity determining regions (HCDRs) consisting of the amino acid sequence of SEQ ID NO: 29, and 3 light chain variable region- contained complementarity determining regions (LCDRs) consisting of the amino acid sequence of SEQ ID NO: 33; or wherein the antigen binding region that specifically binds PD-1 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 30; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 31; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 32; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 34; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 35; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:

36.

6. The immunoconjugate of claim 5, wherein the antigen binding region that specifically binds PD-1 comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 29 or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and / or the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 33 or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; Optionally, the VH and VL comprise or consist of the amino acid sequences set forth below, respectively: SEQ ID NO: 29 and SEQ ID NO:

33.

7. The immunoconjugate of claim 5 or 6, wherein the antigen binding region that specifically binds PD-1 is a Fab fragment comprising a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain comprises VH and CH1, and the Fab light chain comprises VL and CL; Optionally, the CH1 is or is from a CH1 of IgGl, IgG2, IgG3 or IgG4, preferably a CH1 of IgGl, for example the CH1 (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 28; or the CL is or is from a Kappa light chain constant region or a Lambda light chain constant region, for example a human Kappa light chain constant region or a human Lambda light chain constant region, for example the CL (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 37; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO:

37.

8. The immunoconjugate of claim 7, wherein the Fab heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 43; and / or the Fab light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 14; Optionally, wherein the Fab heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 43, and the Fab light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:

14.

9. The immunoconjugate of any one of claims 5-8, wherein the immunoconjugate comprises 1 IL-12 mutein, and 1 or 2 antigen binding domains that specifically bind to PD-1.

10. The immunoconjugate of any one of claims 5-9, wherein the immunoconjugate further comprises a first Fc region linked to an antigen binding domain that specifically binds to PD-1 and a second Fc region linked to an IL-12 mutein; Optionally, the Fc region is or is from a human IgG Fc, for example, a human IgGl Fc, a human IgG2 Fc, a human IgG3 Fc or a human IgG4 Fc, for example the Fc comprises or consists of the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence that is at least 90% identical, for example 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 26; Optionally, the first and second Fc regions each comprise a mutation facilitating heterodimerization thereof, e.g. a hole mutation and a knob mutation, e.g. the first Fc region comprises a hole mutation and the second Fc region comprises a knob mutation, or vice versa; e.g. the knob mutation is S354C and T366W, and the hole mutation is Y349C, T366S, L368A and Y407V. Optionally, the Fc region further comprises a mutation reducing Fc fragment binding to FcyR, e.g. a L234A / L235A mutation.

11. The immunoconjugate of claim 10, wherein the Fc region comprising a knob mutation comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 1 and comprises the mutations S354C and T366W, and optionally L234A / L235A mutations; and the Fc region comprising a hole mutation comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 2 and comprises the mutations Y349C, T366S, L368A and Y407V, and optionally L234A / L235A mutations; Optionally, the Fc region comprising a knob mutation comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 and the Fc region comprising a hole mutation comprises or consists of the amino acid sequence set forth in SEQ ID NO:

2.

12. The immunoconjugate of any one of claims 5-11, comprising a first heavy chain: Fab heavy chain 1 that specifically binds PD-1 - linker 3 - Fab heavy chain 2 that specifically binds PD-1 - first Fc region; a first light chain: Fab light chain 1 that specifically binds PD-1 ; a second light chain: Fab light chain 2 that specifically binds PD-1 ; a second heavy chain: IL-12 mutein - linker 1 - second Fc region; wherein Fab heavy chain 1 and Fab light chain 1 form a first Fab fragment that specifically binds PD-1 ; Fab heavy chain 2 and Fab light chain 2 form a second Fab fragment that specifically binds PD-1 ; wherein the IL-12 mutein comprises the IL-12 p40 subunit variant and the IL-12 p35 subunit variant linked via linker 2, e.g. IL-12 p40 subunit variant - linker 2 - IL-12 p35 subunit variant; e.g. the IL-12 mutein is an IL-12 mutein as defined in any one of claims 1-4; wherein the IL-12 mutein comprises the IL-12 p40 subunit variant and the IL-12 p35 subunit variant linked via linker 2, e.g. IL-12 p40 subunit variant - linker 2 - IL-12 p35 subunit variant; e.g. the IL-12 mutein is an IL-12 mutein as defined in any one of claims 1-4; optionally the first Fc region comprises hole mutations, e.g. Y349C / T366S / L368A / Y407V; and / or the second Fc region comprises knob mutations, e.g. S354C and T366W; optionally the first Fc region and / or the second Fc region comprises mutations having reduced binding to Fcy receptors, e.g. L234A / L235A mutations; for example the first Fc region and the second Fc region are Fc regions as defined in claim 10 or 11; optionally linker 1 is an amino acid sequence as set forth in SEQ ID NO: 22, linker 2 is an amino acid sequence as set forth in SEQ ID NO: 42, and / or linker 3 is an amino acid sequence as set forth in SEQ ID NO: 44 or 45; wherein the first Fab fragment and the second Fab fragment are the same or different, preferably the same, e.g. each is a Fab fragment as defined in claim 7 or 8.

13. The immunoconjugate of claim 12, wherein the first heavy chain comprises, consists of or consists essentially of an amino acid sequence as set forth in SEQ ID NO: 13, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto; the second heavy chain comprises, consists of or consists essentially of an amino acid sequence as set forth in SEQ ID NO: 17 or 18 or 20 or 21, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto; and the first light chain and the second light chain each comprise, consist of or consist essentially of an amino acid sequence as set forth in SEQ ID NO: 14, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto.

14. The immunoconjugate of any one of claims 5-11, comprising a first heavy chain: a Fab heavy chain specific for binding PD-1 - a first Fc region; a light chain: a Fab light chain specific for binding PD-1; and a second heavy chain: an IL-12 mutein - linker 1 - a second Fc region; wherein the Fab heavy chain and the Fab light chain form a Fab fragment specific for binding PD-1, e.g. a Fab fragment as defined in claim 7 or 8; the IL-12 mutein comprises an IL-12 p40 subunit variant and an IL-12 p35 subunit variant connected via linker 2, e.g. IL-12 p40 subunit variant - linker 2 - IL-12 p35 subunit variant; for example, the IL-12 mutein is an IL-12 mutein as defined in any one of claims 1-4; the first Fc region and the second Fc region are Fc regions as defined in claim 10 or 11. Optionally, the first Fc region comprises hole mutations, e.g. Y349C / T366S / L368A / Y407V; and / or the second Fc region comprises knob mutations, e.g. S354C and T366W; optionally the first and / or second Fc region comprises mutations with reduced binding to Fcy receptors, e.g. L234A / L235A mutations; e.g. the first and second Fc region are Fc regions as defined in claim 10 or 11. Optionally linker 1 is an amino acid sequence as set forth in SEQ ID NO: 22, and / or linker 2 is an amino acid sequence as set forth in SEQ ID NO:

42.

15. The immunoconjugate of claim 14, wherein the first heavy chain comprises, consists of or consists essentially of an amino acid sequence as set forth in SEQ ID NO: 12, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto; the second heavy chain comprises, consists of or consists essentially of an amino acid sequence as set forth in SEQ ID NO: 17 or 18, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto; and the light chain comprises, consists of or consists essentially of an amino acid sequence as set forth in SEQ ID NO: 14, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto.

16. An isolated nucleic acid encoding the IL-12 mutein of any one of claims 1-4 or the immunoconjugate of any one of claims 5-15.

17. A vector comprising the nucleic acid of claim 16, preferably the vector is an expression vector.

18. A host cell comprising the nucleic acid of claim 16 or the vector of claim 17, preferably the host cell is prokaryotic or eukaryotic, more preferably selected from a yeast cell, a mammalian cell (e.g. a 293 cell or a CHO cell) or other cell suitable for making an antibody or antigen binding fragment thereof.

19. A method of making an IL-12 mutein or an immunoconjugate comprising the same, the method comprising culturing the host cell of claim 18 under conditions suitable for expression of a nucleic acid encoding the IL-12 mutein of any one of claims 1-4 or the immunoconjugate of any one of claims 5-15, optionally isolating the mutein or the immunoconjugate, optionally the method further comprises recovering the mutein or immunoconjugate from the host cell, optionally the mutein or immunoconjugate is purified, e.g. by Protein A purification.

20. A pharmaceutical composition comprising the immunoconjugate of any one of claims 5-15, and optionally a pharmaceutically acceptable excipient.

21. A pharmaceutical combination comprising the immunoconjugate of any one of claims 5-15, and one or more other therapeutic agents, e.g., various therapeutic agents for treating a tumor, e.g., a chemotherapeutic agent, an angiogenesis inhibitor, a cytokine, a cytotoxic agent, another antibody, a small molecule drug, or an immunomodulatory agent (e.g., an immune checkpoint inhibitor or agonist).

22. A method of treating a tumor, e.g., a cancer, in an individual, the method comprising administering to the subject an effective amount of the immunoconjugate of any one of claims 5-15 or the pharmaceutical composition of claim 20 or the pharmaceutical combination of claim 21; optionally, wherein the tumor is associated with abnormal expression or activity of PD-1 in the individual having the tumor, e.g., elevated PD-1 expression in immune cells, e.g., activated T cells, of the individual, e.g., as compared to a healthy subject; or the tumor is associated with abnormal expression or activity of PD-L1 and / or PD-L2 in the individual having the tumor, e.g., elevated PD-L1 and / or PD-L2 protein or nucleic acid levels in the tumor cells, e.g., as compared to cells of a corresponding tissue of a healthy subject, or as compared to healthy cells proximal to the tumor cells.

23. The method of claim 22, wherein the tumor is selected from the group consisting of intestinal cancer, breast cancer, lung cancer, melanoma, or pancreatic cancer.

24. The method of claim 22 or 23, wherein the method further comprises administering one or more other therapies, e.g., treatment modalities and / or other therapeutic agents, e.g., the therapeutic agent is selected from the group consisting of various therapeutic agents for treating a tumor, e.g., a chemotherapeutic agent, an angiogenesis inhibitor, a cytokine, a cytotoxic agent, another antibody, a small molecule drug, or an immunomodulatory agent (e.g., an immune checkpoint inhibitor or agonist).

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