Multifunctional molecules directed against CD28

Abstract

The present invention provides multifunctional molecules comprising an agonistic anti-CD28 binding domain covalently linked to another binding moiety that binds to a target specifically expressed on the surface of a T cell and uses thereof.

Description

Technical Field

[0001] The present invention relates to the field of medicine, in particular to immunotherapy. The present invention provides a multifunctional molecule comprising a single anti-CD28 binding domain covalently linked to a binding moiety that binds to a target specifically expressed on the surface of an immune cell. Background Art

[0002] The ability of T cells to recognize and kill their cell targets depends on a set of coordinated interactions. The main example of these instructions is that the T cell receptor (TCR) complex recognizes and binds to target cells. Other interactions such as CD28 can further promote T cell activation. T cells present CD28 on their surface, which can provide costimulatory signals to enhance activation via the TCR complex. When T cells recognize their target cells via their TCR complexes, and then participate in costimulatory signals via CD28 bound to their target cells, T cell activation is enhanced.

[0003] Agonistic anti-CD28 mAbs can be used for sustained ex vivo expansion of cultured T cells. However, in a phase I clinical trial testing systemic superagonistic anti-CD28 mAbs, the use of antibodies against CD28 was discouraged due to a series of acute and serious adverse events (HGnig, Nature Reviews Immunology. 2012; 12: 317-318). Local or targeted use of anti-CD28 mAbs can be used to promote anti-tumor immunity with lower risks (Jung et al., Int J Cancer. 2001 Jan 15; 91 (2): 225-30).

[0004] Therefore, there is a need to develop improved agents for safe immunotherapy targeting human CD28, particularly for the treatment of cancer.

[0005] The core of the therapy studied in the prior art is to reduce nonspecific targeting, more specifically by preparing bispecific antibodies against CD28 and tumor-specific antigens (such as PD-L1). It can combine with the co-stimulatory effect of CD28 and block the tumor-targeted checkpoint inhibitory effect of PD-1 / PD-L1 to achieve direct targeting of tumors. Examples of such antibodies can be found in WO2022 / 094299 and WO2022 / 081886. SUMMARY OF THE INVENTION

[0007] The inventors have provided for the first time a multifunctional molecule capable of binding on one side to CD28 and on the other side to another target specifically expressed on human T cells, in particular PD-1. Surprisingly, this multifunctional molecule does not cause the expected problems despite the combination of a CD28 agonist and another T cell-specific antigen binding domain, even though this combination would initially appear to be harmful based on previous experimental results using anti-CD28 superagonists.

[0008] Furthermore, this multifunctional molecule exhibits both cis-targeting and cis-activation capabilities. In fact, it binds to single T cells via both binding domains, resulting in a potent but controlled stimulation of T cells without any risk of overstimulation.

[0009] Furthermore, despite its lack of tumor-specific binding ability, it is able to target T cells located within or near tumors, thereby avoiding systemic activation of T cells that do not have any specificity for tumors.

[0010] The molecules of the invention are particularly useful for the prevention and / or treatment of various diseases, in particular cancer.

[0011] Accordingly, the inventors provide multifunctional molecules for a variety of therapeutic applications, in particular for the treatment of cancer, comprising a single anti-CD28 binding domain covalently linked to a binding moiety that binds to a target specifically expressed on the surface of a T cell.

[0012] The multifunctional molecules of the present invention can bind to T cells in cis. This cis targeting ability specifically activates T cells expressing CD28 and another T-specific antigen (such as PD-1) and does not activate naive nonspecific T cells, thereby avoiding nonspecific activation and limiting the toxicity of the molecules of the present invention.

[0013] Additionally, the multifunctional molecules of the present invention do not exhibit transactivation, ie, co-targeting of tumor cells and T cells, avoiding potential undesirable side effects.

[0014] Thus, the multifunctional molecules of the present invention achieve the goal of providing multifunctional molecules that are effective in treating a variety of disorders, including cancer, but with little or no side effects.

[0015] In a first aspect, the present invention relates to a multifunctional molecule comprising or essentially consisting of:

[0016] (a) a first binding moiety that binds to a target specifically expressed on the surface of T cells, the target being selected from the group consisting of PD-1, VISTA, CTLA-4, BTLA, TIGIT, CD160, LAG3 and TIM3; and

[0017] (b) a second binding moiety which has an agonistic effect on CD28 and is an anti-CD28 antigen binding domain comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) and optionally a heavy chain constant domain (CH1) and a light chain constant domain (CL), wherein:

[0018] (i) the heavy chain variable domain (VH) comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, and

[0019] (ii) a light chain variable domain (VL) comprising complementarity determining regions (CDRs) LCDR1, LCDR2 and LCDR3,

[0020] in:

[0021] - a heavy chain CDR1 (HCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 30, optionally with one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof;

[0022] - a heavy chain CDR2 (HCDR2) comprising or consisting of the amino acid sequence of SEQ ID NO: 31, optionally with one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof;

[0023] - a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 32; optionally having one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof;

[0024] - the light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 33, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof;

[0025] - the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 34, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; and

[0026] - the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 35, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof;

[0027] The multifunctional molecule comprises a single anti-CD28 antigen binding domain.

[0028] Preferably, the target specifically expressed on the surface of T cells is PD-1.

[0029] In some embodiments, the first binding moiety is a first antigen binding domain comprising VH and VL and optionally a light chain constant domain (CL) and a heavy chain constant domain (CH1), and wherein the first antigen binding domain is linked to the N-terminus of the first Fc chain, and the molecule further comprises a second Fc chain that forms an Fc domain with the first Fc chain.

[0030] Preferably, the anti-CD28 antigen binding protein comprises VH and VL, and optionally CL and CH1, preferably comprises:

[0031] (i) a heavy chain variable domain (VH) comprising HCDR1, HCDR2 and HCDR3, and

[0032] (ii) a light chain variable domain (VL) comprising LCDR1, LCDR2 and LCDR3,

[0033] in:

[0034] - heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 30;

[0035] - heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 31;

[0036] - heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 32;

[0037] - light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 33;

[0038] - the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 34,

[0039] - light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 35;

[0040] Even more preferably comprises (a) VH comprising or consisting of the amino acid sequence of SEQ ID NO: 44, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; and (b) VL comprising or consisting of the amino acid sequence of SEQ ID NO: 45, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0041] In particular, the anti-CD28 antigen binding domain is covalently linked to: (i) the C-terminus of CL of the first binding moiety, (ii) the C-terminus of CH1 of the first binding moiety, (iii) the N-terminus of VH of the first binding moiety, or (iii) the N-terminus of VL of the first binding moiety, optionally via a peptide linker.

[0042] In some embodiments, the molecule comprises a first Fc chain and a second Fc chain that together form an Fc domain, and the anti-CD28 antigen binding domain is covalently linked to the C-terminus of one of the Fc chains, preferably the first Fc chain that is covalently linked to the first antigen binding domain at its N-terminus, optionally via a peptide linker.

[0043] In some embodiments, the molecule comprises a first Fc chain and a second Fc chain that together form an Fc domain, the anti-CD28 antigen binding domain is covalently linked via its C-terminus to the N-terminus of the second Fc chain, and the first antigen binding domain is covalently linked via its C-terminus to the N-terminus of the first Fc chain, optionally via a peptide linker.

[0044] Preferably, the anti-CD28 antigen binding domain is Fab, CrossMAb or scFv, preferably scFv.

[0045] In particular, in the multifunctional molecules according to the invention:

[0046] - the anti-CD28 antigen binding domain is a scFv, with VH connected to VL from N-terminus to C-terminus, preferably via a peptide linker; or

[0047] - The anti-CD28 antigen binding domain is a scFv, with VL and VH connected from N-terminus to C-terminus, preferably via a peptide linker.

[0048] In some embodiments, the anti-CD28 antigen binding domain is a Fab or a CrossMAb.

[0049] In particular, in the anti-CD28 antigen binding domain, (i) the heavy chain variable domain (VH) further comprises heavy chain variable region framework regions (HFRs) HFR1, HFR2, HFR3 and HFR4, which comprise or consist of the amino acid sequences of SEQ ID NOs: 36, 37, 38 and 39, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, and

[0050] (ii) the light chain variable domain (VL) further comprises framework regions (LFR) LFR1, LFR2, LFR3 and LFR4, which comprise or consist of the amino acid sequences of SEQ ID NOs: 40, 41, 42 and 43, respectively, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof.

[0051] More particularly, the anti-CD28 antigen binding domain comprises: (a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 44, 83, 84, 85 and 88, optionally having one, two or three amino acid modifications selected from substitutions, additions, deletions and any combination thereof, preferably substitutions; and (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 86, 87, 89 and 90, optionally having one, two or three amino acid modifications selected from substitutions, additions, deletions and any combination thereof, preferably substitutions.

[0052] Preferably, the anti-CD28 antigen binding domain comprises: (a) a VH comprising or consisting of an amino acid sequence of SEQ ID NO: 44, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, preferably in the framework regions; and (b) a VL comprising or consisting of an amino acid sequence of SEQ ID NO: 45, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, preferably in the framework regions.

[0053] In some embodiments, in the multifunctional molecule of the present invention, the first binding moiety is or is derived from an anti-PD-1 antibody selected from the group consisting of Pembrolizumab, Nivolumab, Pidilizumab, Cemiplimab, Camrelizumab, AUNP12, AMP-224, AGEN-2034, Tisleizumab, PDR001, MK -3477, PF-06801591, JNJ-63723283, genolimzumab, LZM-009, BCD-100, SHR-1201, BAT-1306, AK-103, MEDI-0680, JS001, BI-754091, CBT-501, INCSHR1210, TSR-042, GLS-010, AM-0001, STI-1110, MGA012 or IBI308, preferably pembrolizumab or nivolumab.

[0054] In some embodiments, the first binding moiety is an anti-PD-1 antigen binding domain, preferably comprising:

[0055] (i) a VH comprising HCDR1, HCDR2 and HCDR3, and (ii) a VL comprising LCDR1, LCDR2 and LCDR3,

[0056] in:

[0057] - heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 1;

[0058] - heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 2;

[0059] - heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 3;

[0060] - light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 4;

[0061] - the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 5, and

[0062] - the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 6,

[0063] Preferably, the anti-PD-1 antigen binding domain is F(ab')2, Fab or CrossMAb.

[0064] Preferably, the first binding moiety is an anti-PD-1 antigen binding domain, preferably comprising: (a) VH, which comprises or consists of the amino acid sequence of SEQ ID NO: 15, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; preferably in the framework region; and (b) VL, which comprises or consists of the amino acid sequence of SEQ ID NO: 16, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, preferably in the framework region.

[0065] Preferably, the anti-PD-1 antigen binding domain is F(ab')2, Fab or CrossMAb.

[0066] In some embodiments, the first binding moiety is an anti-PD-1 antigen binding domain comprising:

[0067] a) (i) a VH comprising HCDR1, HCDR2 and HCDR3, and (ii) a VL comprising LCDR1, LCDR2 and LCDR3, wherein: the heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 65; the heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 66; the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 67; the light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 68; the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 69, and the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 70; or

[0068] b) i) a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 71; and ii) a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 72; or

[0069] c) (i) a VH comprising HCDR1, HCDR2 and HCDR3, and (ii) a VL comprising LCDR1, LCDR2 and LCDR3, wherein: the heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 73; the heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 74; the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 75; the light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 76; the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 77, and the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 78; or

[0070] d) i) a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 79; and ii) a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 80.

[0071] In a second aspect, the present invention relates to an isolated nucleic acid sequence or an isolated set of nucleic acid sequences encoding a multifunctional molecule according to the present invention, or a vector comprising said isolated nucleic acid sequence or an isolated set of nucleic acid sequences.

[0072] In a third aspect, the present invention relates to a host cell comprising the vector or the isolated nucleic acid or the isolated set of nucleic acid molecules of the present invention.

[0073] In a fourth aspect, the present invention relates to a method for producing a multifunctional molecule according to the present invention, comprising the steps of culturing a host cell according to the present invention and isolating the multifunctional molecule.

[0074] In a fifth aspect, the present invention relates to a pharmaceutical composition comprising a multifunctional molecule according to the present invention, a nucleic acid or a group of nucleic acid molecules according to the present invention or a vector according to the present invention or a host cell according to the present invention and a pharmaceutically acceptable carrier.

[0075] In particular, the pharmaceutical composition, the multifunctional molecule, the nucleic acid, the set of nucleic acid molecules, the vector, the host cell according to the present invention are used as a medicament.

[0076] In particular, the pharmaceutical composition, multifunctional molecule, nucleic acid, nucleic acid molecule group, vector, host cell according to the present invention is used for treating cancer or treating infectious diseases.

[0077] Preferably, the disease is a cancer selected from the group consisting of brain cancer, carcinoma, cervical cancer, colorectal cancer, esophageal cancer, stomach cancer, gastrointestinal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, lymphoma, glioma, mesothelioma, melanoma, gastric cancer, urethral cancer, environmentally induced cancers, and any combination of said cancers.

[0078] Alternatively, the disease is an infectious disease caused by a virus selected from the group consisting of human immunodeficiency virus, hepatitis virus, herpes virus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, human T-lymphotropic virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus.

[0079] In some embodiments, the pharmaceutical composition, multifunctional molecule, nucleic acid or set of nucleic acid molecules, vector or host cell according to the invention is used in combination with radiotherapy or with another therapeutic agent, in particular a chemotherapeutic agent or an anti-infective agent.

[0080] In particular, the additional therapeutic agent is selected from the group consisting of a chemotherapeutic agent, an anti-infective agent, an alkylating agent, an angiogenesis inhibitor, an antibody, an antimetabolite, an antimitotic agent, an antiproliferative agent, an antiviral agent, an Aurora kinase inhibitor, an apoptosis promoter, a death receptor pathway activator, a Bcr-Abl kinase inhibitor, a bispecific T cell engager antibody drug conjugate, a Bruton's tyrosine kinase (BTK) inhibitor, a cyclin-dependent kinase inhibitor, a cell cycle inhibitor, a cyclooxygenase-2 inhibitor, a leukemia virus oncogene homolog receptor inhibitor, a growth factor inhibitor, a heat shock protein-90 inhibitor, a histone deacetylase inhibitor, a hormone therapy, an apoptosis protein inhibitor, an antibiotic, a kinase inhibitor Agents, kinesin inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNA, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, nonsteroidal anti-inflammatory drugs, poly ADP ribose polymerase inhibitors, platinum chemotherapeutic agents, polo-like kinase inhibitors, phosphoinositide-3 kinase inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoids, small inhibitory ribonucleic acids (siRNA), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, peptide vaccines, cancer vaccines, epitopes or new epitopes from tumor antigens, and combinations of one or more of these agents.

[0081] Finally, the present invention relates to a method for treating a disease, such as cancer or an infectious disease, in a subject in need thereof, comprising administering the multifunctional molecule or pharmaceutical composition of the present invention, preferably in an effective amount.

[0082] The present invention also relates to the use of the multifunctional molecule or pharmaceutical composition according to the present invention in the preparation of a medicament for treating a disease such as cancer or an infectious disease. DETAILED DESCRIPTION OF THE INVENTION

[0084] definition

[0085] In order that the present invention may be more readily understood, certain terms are defined below. Additional definitions are set forth throughout the detailed description.

[0086] Unless otherwise defined, all technical terms, symbols and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art to which the present invention belongs. In some cases, for clarity and / or ease of reference, terms with commonly understood meanings are defined herein, and the inclusion of such definitions herein should not necessarily be interpreted as representing differences from those commonly understood in the art. The techniques and procedures described or cited herein are generally well known to those skilled in the art and are commonly employed using conventional methods.

[0087] As used herein, the term "antibody" describes a class of immunoglobulin molecules and is used in its broadest sense. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, that is, molecules containing antigen binding sites. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ and μ, respectively. Unless otherwise specifically stated, the term "antibody" includes complete immunoglobulins and "antibody fragments" or "antigen binding fragments", such as Fab, Fab', F(ab')2, Fv, single chain (scFv), CrossMAb, molecules comprising antibody portions, double antibodies, linear antibodies, single chain antibodies and any other modified configurations of immunoglobulin molecules comprising antigen recognition sites of desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies. Preferably, the term antibody refers to a humanized antibody. In terms of structure, an antibody may have a heavy (H) chain and a light (L) chain that are interconnected by a disulfide bond. There are two types of light chains: lambda (λ) and kappa (κ). Each heavy chain and light chain contains a constant region and a variable region (or "domain"). Both the light chain and the heavy chain variable region contain a "framework" region that is interrupted by three hypervariable regions (also referred to as "complementarity determining regions" or "CDRs"). The scope of the framework region and CDR has been determined (see, Kabat et al., Sequences of Proteins of Immunological Interest, and US Department of Health and Human Services, 1991, which are incorporated herein by reference). Preferably, CDR is defined according to Kabat or IGMT method, preferably by Kabat method. The framework region plays a role in forming a scaffold that positions the CDR in the correct direction by non-covalent interactions between chains. CDR is primarily responsible for binding to the epitope of the antigen. The CDRs of each chain are usually referred to as "complementarity determining region 1" or "CDR1", "CDR2" and "CDR3", and are numbered sequentially starting from the N-terminus. The VL and VH domains of antibodies according to the present invention may include four framework regions or "FRs", which are respectively referred to in the art and herein as "framework region 1" or "FR1", "FR2", "FR3" and "FR4". These framework regions and complementarity determining regions are preferably operably linked in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (from amino terminus to carboxyl terminus).

[0088] "CrossMab technology" is based on the crossover of domains between heavy and light chains, thereby creating different domain arrangements for heavy and light chains of different specificities. WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, WO2009 / 080254 and Schaefer, W. et al, PNAS, 108 (201 1) 11187-1 191 (the disclosures of which are incorporated herein by reference) relate to bivalent bispecific IgG antibodies with domain crossover. In CrossMab, (i) constant domains CL and CH1 replace each other, and / or (ii) constant domains VL and VH replace each other. Accordingly, the antigen binding domain comprises a chain with VH and CL, and another chain with VL and CH1. As used herein, the term "CrossMAb" refers to an antigen binding domain with a reversal of the CL and CH1 domains, particularly in one binding arm of an antibody. Thus, such a binding domain comprises a VH domain connected to the CL domain and a VL domain connected to the CH1 domain. Such a configuration reduces the formation of byproducts caused by the mispairing of the light chain of a first binding domain that specifically binds to a first antigen with the wrong heavy chain of a second binding domain that specifically binds to a second antigen (when compared to a method without such a CL-CH1 domain exchange). CrossMAbs typically have a Fab conformation, but have the above-mentioned CL and CH1 reversals. For example, they are described in WO 2009 / 080253 and Schaefer, W. et al, PNAS, 108 (2011) 11187-1191, the disclosures of which are incorporated herein by reference.

[0089] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in the antibody conformation. The heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2, and CH3. The VH region can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). VH specifically comprises three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of the antibody heavy chain are commonly referred to as "HCDR1", "HCDR2", and "HCDR3". The framework regions of the antibody heavy chain are commonly referred to as "HFR1", "HFR2", "HFR3", and "HFR4".

[0090] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in the antibody conformation; kappa and lambda light chains refer to the two major antibody light chain isotypes. The light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). The light chain constant region comprises one domain, CL. The VL region can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). VL specifically comprises three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of an antibody light chain are commonly referred to as "LCDR1", "LCDR2", and "LCDR3". The framework regions of an antibody light chain are commonly referred to as "LFR1", "LFR2", "LFR3", and "LFR4".

[0091] As used herein, an "antigen-binding fragment" or "antigen-binding domain" of an antibody refers to a portion of an antibody, i.e., a molecule corresponding to a portion of the antibody structure of the present invention, which exhibits antigen-binding ability to a specific antigen, possibly in its native form; such fragments particularly exhibit the same or substantially the same antigen-binding specificity to the antigen compared to the antigen-binding specificity of the corresponding four-chain antibody. Advantageously, the antigen-binding fragment has a similar binding affinity to the corresponding four-chain antibody. However, antigen-binding fragments with reduced antigen-binding affinity relative to the corresponding four-chain antibody are also included in the present invention. Antigen binding ability can be determined by measuring the affinity between the antibody and the target fragment. These antigen-binding fragments may also be referred to as "functional fragments" of antibodies. The antigen-binding fragment of an antibody is a fragment comprising its hypervariable domain (complementarity determining region) called CDR or a portion thereof. For example, such an antigen-binding domain may be a Fab, scFv or CrossMab.

[0092] As used herein, the term "humanized antibody" refers to an antibody in which a CDR sequence derived from another mammalian species (e.g., mouse) germline is transplanted to a human framework sequence (e.g., a chimeric antibody containing a minimal sequence derived from a non-human antibody). A "humanized form" of an antibody, e.g., a non-human antibody, also refers to an antibody that has undergone humanization. A humanized antibody is typically a human immunoglobulin (recipient antibody), in which residues from one or more CDRs are replaced by residues from at least one CDR of a non-human antibody (donor antibody), while maintaining the desired specificity, affinity, and ability of the original antibody. Additional framework region modifications may be performed within the human framework sequence. Preferably, the humanized antibody has a T20 humanization score greater than 80%, 85%, or 90%. For example, the "humanization" of an antibody can be measured using the T20 score analyzer to quantify the humanization of an antibody variable region, as described in Gao SH, Huang K, TuH, Adler A S. BMC Biotechnology. 2013: 13:55, or via a web-based tool using the T20 Cutoff Human Databases: http: / / abAnalyzer.lakepharma.com to calculate the T20 score for an antibody sequence.

[0093] "Chimeric antibodies" refer to antibodies made by combining genetic material from a non-human source (preferably, a mouse) with human genetic material. Such antibodies are derived from human and non-human antibodies connected by a chimeric region. Chimeric antibodies typically contain constant domains from humans and variable domains from other mammalian species, and when they are used for therapeutic treatments, they can reduce the risk of reactions with foreign antibodies from non-human animals.

[0094] As used herein, the terms "crystallizable fragment region", "Fc region" or "Fc domain" are interchangeable and refer to the tail region of an antibody that interacts with a cell surface receptor called an Fc receptor. The Fc region or domain typically comprises two (optionally identical) domains derived from the second and third constant domains (i.e., CH2 and CH3 domains) of the two heavy chains of an antibody. The portion of the Fc domain refers to the CH2 or CH3 domain. Optionally, the Fc region or domain may optionally comprise all or part of the hinge region between CH1 and CH2. Thus, the Fc domain may comprise a hinge, a CH2 domain, and a CH3 domain. Optionally, the Fc domain is an Fc domain from IgG1, IgG2, IgG3, or IgG4, optionally having an IgG1 hinge-CH2-CH3 and an IgG4 hinge-CH2-CH3.

[0095] With respect to the "binding" ability of the binding moieties described herein, the term "bind" or "binding" refers to antibodies including antibody fragments and derivatives that recognize and contact another peptide, polypeptide, protein or molecule. The terms "specifically bind", "specifically bind to", "specific for", "selectively bind to" and "selectively for" a particular target refer to an antigen binding domain that recognizes and binds to a particular target but does not substantially recognize or bind to other molecules in a sample. For example, an antibody that specifically (or preferentially) binds to an antigen is an antibody that binds to the antigen, for example, with greater affinity, avidity, more readily and / or for a longer duration than it binds to other molecules. Preferably, the term "specifically binds" refers to an antibody (or antigen binding domain) that binds to the antigen with a specificity equal to or less than 10 -7 In some aspects, the antibody has a binding affinity of equal to or less than 10 -8 M, 10 -9 M or 10 -10 Affinity binding of M.

[0096] "Amino acid change" or "amino acid modification" refers to a change in the amino acid sequence of a polypeptide in this article. "Amino acid modification" includes replacement, insertion and / or deletion of a polypeptide sequence. "Amino acid substitution" or "substitution" herein refers to replacing the amino acid with another amino acid at a specific position in the parent peptide sequence. "Amino acid insertion" or "insertion" refers to adding an amino acid at a specific position in the parent polypeptide sequence. "Amino acid deletion" or "deletion" refers to removing an amino acid at a specific position in the parent polypeptide sequence. Amino acid substitutions can be conservative. Conservative substitutions replace a given amino acid residue with another residue having a side chain ("R-group") of similar chemical properties (e.g., charge, volume and / or hydrophobicity). As used herein, "amino acid position" or "amino acid position numbering" are used interchangeably and refer to the position of a specific amino acid in an amino acid sequence, usually specified by a one-letter code for amino acids. The first amino acid in an amino acid sequence (i.e., starting from the N-terminus) should be considered to have position 1.

[0097] Conservative substitutions are substitutions of a given amino acid residue with another residue having a side chain ("R-group") of similar chemical properties (e.g., charge, volume, and / or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of the protein. Conservative substitutions and corresponding rules are well described in the prior art. For example, conservative substitutions can be defined by substitutions within the amino acid groups shown in the following table:

[0098] Table A - Amino Acid Residues

[0099]

[0100] Table B - Alternative conservative amino acid residue substitution groups

[0101]

[0102] Table C – Further alternative physical and functional classifications of amino acid residues

[0103]

[0104] As used herein, "sequence identity" between two sequences is described by the parameters "sequence identity", "sequence similarity" or "sequence homology". For the purposes of the present invention, the "percent identity" between two sequences (A) and (B) is determined by comparing two aligned sequences in an optimized manner and by a comparison window. The sequence alignment can be performed by methods well known in the art, such as using the Needleman-Wunsch global alignment algorithm. Protein analysis software uses measurements of similarity assigned to various substitutions, deletions and other modifications (including conservative amino acid substitutions) to match similar sequences. Once a total comparison is obtained, the percentage of identity can be obtained by dividing the total number of aligned identical amino acid residues by the total number of residues contained in the longest sequence between sequences (A) and (B). Sequence identity is typically determined using sequence analysis software. For comparing two amino acid sequences, one can use, for example, the tool "Emboss needle" for pairwise alignment of proteins provided by EMBL-EBI and available at the following website: www.ebi.ac.uk / Tools / services / web / toolform.ebi?tool=emboss_needle&context=protein, for example using the default settings: (i) Matrix: BLOSUM62, (ii) Gap Opening: 10, (iii) Gap Extension: 0.5, (iv) Output Format: Pairs, (v) Terminal Gap Penalty: False, (vi) Terminal Gap Opening: 10, (vii) Terminal Gap Extension: 0.5.

[0105] Alternatively, sequence identity can also be determined typically using the sequence analysis software Clustal Omega, using the HHalign algorithm with default settings as its core alignment engine. The algorithm is described in Söding, J. (2005) 'Proteinhomology detection by HMM–HMM comparison'. Bioinformatics 21, 951-960, with default settings.

[0106] "Eu numbering" (also called the Eu index) refers to the antibody numbering system (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda), which is based on the sequence numbering of the first sequenced human IgG1 (the Eu antibody; Edelman, et al., 1969, Proc Natl Acad Sci USA 63:78-85).

[0107] As used herein, the terms "derive from" and "derived from" refer to compounds that have a structure derived from a parent compound or protein and whose structure is sufficiently similar to the structure disclosed herein and, based on such similarity, one skilled in the art would expect that it would exhibit the same or similar properties, activities and utilities as the claimed compound.

[0108] As used herein, "pharmaceutical composition" refers to a preparation of one or more active agents (e.g., comprising a multifunctional molecule according to the present invention) and optional other chemical components (e.g., physiologically suitable carriers and excipients). The purpose of the pharmaceutical composition is to promote the administration of the active agent to an organism. The composition of the present invention can be in the form of a conventional route suitable for any administration or use. In one aspect, "composition" generally means an active agent (e.g., a compound or composition) and a naturally occurring or non-naturally occurring inert (e.g., a detectable agent or marker) or active carrier (e.g., an adjuvant, a diluent, an adhesive, a stabilizer, a buffer, a salt, a lipophilic solvent, a preservative, an adjuvant, etc., and includes a pharmaceutically acceptable carrier). "Acceptable vehicle" or "acceptable carrier" as mentioned herein is any known compound or combination of compounds known to those skilled in the art that can be used to prepare a pharmaceutical composition.

[0109] As used herein, "effective amount" or "therapeutically effective amount" refers to the amount of an active agent required, alone or in combination with one or more other active agents, to impart a therapeutic effect to a subject, e.g., the amount of an active agent required to treat a targeted disease or condition or produce a desired effect. An "effective amount" will vary depending on the agent, the disease and its severity, the characteristics of the subject to be treated (including age, physical condition, size, sex, and weight), the duration of treatment, the nature of concurrent therapy (if any), the specific route of administration, and similar factors within the knowledge and expertise of a health practitioner. These factors are well known to those of ordinary skill in the art and can be resolved only by routine experimentation. It is generally preferred to use the maximum dose of a single component or a combination thereof, i.e., the highest safe dose based on reasonable medical judgment.

[0110] As used herein, the term "drug" refers to any substance or composition having curative or preventive properties against a condition or disease.

[0111] The term "treatment" refers to any action intended to improve the patient's health, for example, treatment, prevention, prophylaxis and delay of a disease or the symptoms of a disease. It specifies the curative treatment and / or preventive treatment of a disease. Curative treatment is defined as a treatment that results in a cure or a treatment that alleviates, improves and / or eliminates, reduces and / or stabilizes a disease or the symptoms of a disease or the pain caused directly or indirectly by it. Preventive treatment includes treatment that results in disease prevention and treatment that reduces and / or delays the progression and / or morbidity of a disease or the risk of its occurrence. In some aspects, this term refers to the improvement or eradication of a disease, a disorder, an infection or the symptoms associated therewith. In other aspects, the term refers to minimizing the spread or deterioration of cancer. Treatment according to the present invention does not necessarily mean 100% or complete treatment. On the contrary, there are different degrees of treatment, and those skilled in the art will recognize that the treatment has potential benefits or therapeutic effects. Preferably, the term "treatment" refers to the application or administration of a composition comprising one or more active agents to a subject suffering from a disorder / disease.

[0112] As used herein, the term "disorder" or "disease" refers to an organ, part, structure or system of the body that does not function properly due to genetic or developmental errors, infections, poisons, nutritional deficiencies or imbalances, toxicity or the effects of adverse environmental factors. Preferably, these terms refer to health conditions or diseases, e.g., diseases that disrupt normal physical or mental function. More preferably, the term "disorder" refers to immune and / or inflammatory diseases affecting animals and / or humans, such as cancer.

[0113] As used herein, "immune cells" refer to cells involved in innate and adaptive immunity, such as white blood cells (leukocytes), which are derived from hematopoietic stem cells (HSC) produced in the bone marrow, lymphocytes (T cells, B cells, natural killer (NK) cells and natural killer T cells (NKT) and myeloid-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). In particular, immune cells can be selected from a non-exhaustive list including B cells, T cells, in particular CD4+ T cells and CD8+ T cells, NK cells, NKT cells, APC cells, dendritic cells and monocytes. "T cells" as used herein include, for example, CD4+ T cells, CD8+ T cells, T helper type 1 T cells, T helper type 2 T cells, T helper type 17 T cells and suppressor T cells.

[0114] As used herein, the term "T effector cell", "T eff" or "effector cell" describes a group of immune cells that include several T cell types that actively respond to stimulation (such as co-stimulation). It particularly includes T cells that have the function of eliminating antigens (e.g., by producing cytokines that regulate the activation of other cells or by cytotoxic activity). It mainly includes CD4+, CD8+, cytotoxic T cells and helper T cells (Th1 and Th2).

[0115] As used herein, the term "regulatory T cells", "Treg cells" or "T regs" refers to a subpopulation of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune diseases. Tregs have immunosuppressive effects and typically inhibit or downregulate the induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FOXP3, and CD25 and are believed to be derived from the same lineage as naive CD4 cells.

[0116] The term "exhausted T cells" refers to a population of T cells that are in a state of dysfunction (i.e., "exhaustion"). T cell exhaustion is characterized by progressive loss of function, changes in transcriptional profiles, and persistent expression of inhibitory receptors. Exhausted T cells lose their cytokine production capacity, their high proliferation capacity, and their cytotoxic potential, which ultimately leads to their loss. Exhausted T cells typically show higher levels of CD43, CD69, and inhibitory receptors, as well as lower expression of CD62L and CD127.

[0117] The term “effector memory stem cell-like T cells” refers to a subset of tumor-reactive intratumoral T cells that have features of exhausted cells and central memory cells, including expression of the checkpoint protein PD-1 and the transcription factor Tcf1. These cells can be referred to as Tcf1 + PD-1 + CD8 +T cells. These cells are present in the tumor microenvironment and are essential for immune control of cancer promoted by immunotherapy. They are essential for maintaining T cell responses during chronic viral infection and cancer, and provide the proliferation burst that occurs after PD-1 immunotherapy. These cells undergo slow self-renewal and produce more terminally differentiated exhausted CD8 T cells. These cells and their characteristics are further defined in the following articles, the disclosures of which are incorporated herein by reference: Siddiqui et al, 2019, Immunity, 50, 195-211; and Jadhav et al, 2019, PNAS, 116, 14113-14118).

[0118] The term "immune response" refers to the actions of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement), which result in the selective injury, destruction, or elimination from the human body of invading pathogens, cells or tissues infected by pathogens, cancer cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.

[0119] As used herein, the term "antagonist" refers to a molecule that blocks or reduces the biological activity or function of another molecule. In particular, the term refers to an antibody or fragment thereof that binds to a cell receptor (e.g., PD-1) such as a reference substance (e.g., PD-L1 and / or PD-L2), preventing it from producing all or part of its usual biological effects (e.g., producing an immunosuppressive microenvironment). The antagonist activity of the humanized antibody according to the present invention can be assessed by competitive ELISA.

[0120] As used herein, the term "agonist" refers to a molecule that induces and / or increases the biological activity, function and / or expression of another molecule. According to a specific embodiment, the agonist induces and / or increases the costimulatory effect on immune cells (e.g., T cells). According to a specific embodiment, the agonist induces and / or increases the signal transduction to immune cells (e.g., T cells). In particular, the term refers to an antibody or fragment thereof that is combined with CD28 and stimulates / induces the costimulatory signal required for T cell action. The agonist activity of the humanized antibody according to the present invention can be evaluated by measuring IL-2 secretion in a T cell bioassay.

[0121] As used herein, the terms "pharmacokinetics" and "PK" can be used interchangeably and refer to the fate of a compound, substance or drug administered to a living body. Pharmacokinetics particularly includes ADME or LADME schemes, which represent release (i.e., release of a substance from a composition), absorption (i.e., entry of a substance into the blood circulation), distribution (i.e., dispersion or dissemination of a substance in vivo), metabolism (i.e., conversion or degradation of a substance) and excretion (i.e., removal or removal of a substance from an organism). The two stages of metabolism and excretion can also be classified as elimination. Those skilled in the art can monitor different pharmacokinetic parameters, such as elimination half-life, elimination constant rate, clearance rate (i.e., the volume of plasma clearance of a drug per unit time), Cmax (maximum serum concentration) and drug exposure (determined by the area under the curve, see Scheff et al, Pharm Res. 2011 May; 28 (5): 1081-9) etc.

[0122] As used herein, the term "isolated" means that the material (e.g., antibody, polypeptide, nucleic acid, etc.) is substantially separated from other materials with which it coexists in nature, or is enriched relative to other materials. In particular, an "isolated" antibody is a molecule that has been identified and separated and / or recovered from a component of its natural environment.

[0123] As used herein, the term "and / or" should be considered as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" should be considered as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, just as if each were set forth individually.

[0124] The terms "a" or "an" may refer to one or more of the elements it modifies (eg, "an agent" may refer to one or more agents) unless the context clearly describes either or more than one of the elements.

[0125] As used herein, the term "about" is related to any and all values ​​(including the lower and upper limits of the numerical range) and refers to any value with an acceptable deviation range of up to + / - 10% (e.g., + / - 0.5%, + / - 1%, + / - 1.5%, + / - 2%, + / - 2.5%, + / - 3%, + / - 3.5%, + / - 4%, + / - 4.5%, + / - 5%, + / - 5.5%, + / - 6%, + / - 6.5%, + / - 7%, + / - 7.5%, + / - 8%, + / - 8.5%, + / - 9%, + / - 9.5%). The term "about" is used at the beginning of a series of values ​​to modify each value (i.e., "about 1, 2, and 3" means about 1, about 2, and about 3). Furthermore, when a list of values ​​is described herein (e.g., about 50%, 60%, 70%, 80%, 85%, or 86%), the list includes all intermediate and fractional values ​​thereof (e.g., 54%, 85.4%).

[0126] As used herein, the term "substantially" in relation to any given biological sequence means that the biological sequence differs from a reference sequence contained in the sequence listing by up to 10% of the length of the biological sequence. In particular, "essentially consisting of" means that the biological sequence consists of this sequence, but may also include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, additions, deletions or mixtures thereof, preferably 1, 2, 3, 4 or 5 substitutions, additions, deletions or mixtures thereof, with the proviso that the biological sequence differs from a reference sequence contained in the sequence listing by up to 10% of the length of the biological sequence.

[0127] Multifunctional molecular structure

[0128] In a first aspect, the present invention relates to a multifunctional molecule comprising:

[0129] (a) a first binding moiety that binds to a target specifically expressed on T cells; and

[0130] (a) a second binding moiety, which is an agonistic anti-CD28 antigen binding domain;

[0131] The multifunctional molecule comprises a single anti-CD28 antigen binding domain.

[0132] The first and second binding moieties target two different targets, making the molecules according to the invention "multifunctional", i.e., able to bind to at least two different targets (e.g., CD28 and a target specifically expressed on the surface of T cells, such as PD-1). The first and second binding moieties target two different targets, making the molecules according to the invention particularly "bifunctional", i.e., able to bind to two different targets (e.g., CD28 and a target specifically expressed on the surface of T cells, such as PD-1).

[0133] The multifunctional molecule may specifically comprise two, three or four binding moieties, but only one binding moiety for CD28. For example, the multifunctional molecule may comprise:

[0134] - a binding moiety that binds to a target specifically expressed on the surface of a T cell and a binding moiety that binds to CD28; or

[0135] - two binding moieties that bind to a target specifically expressed on the surface of a T-cell, said two binding moieties binding to the same or different targets; and one binding moiety that binds to CD28; or

[0136] - three binding moieties that bind to targets specifically expressed on the surface of T cells, the three binding moieties binding to the same or different targets; and one binding moiety that binds to CD28.

[0137] The first and second binding moieties are preferably antigen binding domains, particularly derived from Fab, Fab', F(ab')2, Fv, single chain (scFv), CrossMAb or nanobody (VHH), preferably antigen binding domains of F(ab')2, Fab, CrossMAb or scFV. In the multifunctional molecule, the first and second binding moieties may have the same or different configurations. For example, the first and second binding moieties may both be Fab or scFV. Alternatively, the first and second binding moieties are not both scFv. In a preferred aspect, the anti-CD28 antigen binding moiety is scFv, and the binding moiety that binds to a target specifically expressed on the surface of a T cell is not scFv. For example, the first binding moiety may be F(ab')2, CrossMAb or Fab, and the second binding moiety may be scFv, Fab or CrossMAb.

[0138] In particular, the first binding moiety is F(ab')2, CrossMAb or Fab, and the second binding moiety is scFv.

[0139] The multifunctional molecule preferably comprises: i) a first and a second binding moiety, i) optionally a CH1, ii) optionally a hinge, iii) optionally a CH2 domain and a CH3 domain.

[0140] In the context of IgG antibodies, IgG isotypes each have three heavy chain constant regions (CH). Thus, the "CH" domains in the context of IgG antibodies are as follows: "CH1" refers to the first constant domain (most N-terminal) of the three constant domains of the heavy chain. In particular, it refers to positions 118-215 according to the EU index of Kabat. "Hinge" refers to positions 216-230 according to the EU index of Kabat. "CH2" refers to the second constant domain and in particular to positions 231-340 according to the EU index in Kabat, and "CH3" refers to the third constant domain (most C-terminal) and in particular to positions 341-447 according to the EU index in Kabat. The CH domain may be a naturally occurring CH domain, or a naturally occurring CH domain in which one or more amino acids have been substituted.

[0141] The term "hinge region" refers to a flexible polypeptide comprising amino acids between the CH1 and CH2 domains of an antibody. For the purposes of the present invention, the hinge is structurally defined. As used herein, the IgG1 "hinge region" comprises residues 216-230 according to the EU index in Kabat.

[0142] The first and / or second binding moiety may comprise a CL domain.

[0143] In the context of IgG antibodies, each IgG isotype has a light chain constant domain (CL). A "CL domain" refers to a light chain immunoglobulin constant domain located at the C-terminus of the VL domain. It ranges from about EU index Kabat positions 107-216. The CL domain can be a naturally occurring CL domain, or a naturally occurring CL domain in which one or more amino acids are substituted.

[0144] Such multifunctional molecules preferably comprise a first Fc chain and a second Fc chain that are complementary and together form an Fc domain. The first and second binding moieties may be located on the same or different Fc chains, thereby forming a homodimeric Fc domain or a heterodimeric Fc domain, respectively.

[0145] Therefore, the multifunctional molecule may comprise two binding moieties, preferably two antigen binding domains and an Fc domain. Preferably, the multifunctional molecule of the present invention comprises a first and a second antigen binding domain and an Fc domain. In addition, the multifunctional molecule may further comprise a hinge domain.

[0146] Additionally, the multifunctional molecule may further comprise one or more peptide linkers that allow for linkage between different parts of the multifunctional molecule (eg, between the Fc chain and the binding moiety).

[0147] In an embodiment, the first and second binding moieties are Fabs, such that the multifunctional molecule preferably comprises or consists of:

[0148] (i) a first light chain, which comprises a variable domain (VL) and a constant domain (CL),

[0149] (ii) a first heavy chain, which comprises a variable domain (VH) and a constant domain (CH1),

[0150] The first light chain and the heavy chain form a first binding moiety that binds to a target specifically expressed on the surface of a T cell;

[0151] (iii) a second light chain, which comprises a variable domain (VL) and a constant domain (CL),

[0152] (iv) a second heavy chain comprising a variable domain (VH) and a constant domain (CH1),

[0153] The second light chain and heavy chain form a second binding moiety that binds to CD28.

[0154] Such molecules may further comprise an Fc domain, such that the multifunctional molecule preferably comprises two different Fab domains and an Fc domain. In particular, the multifunctional molecule comprises or consists of:

[0155] (i) a first light chain, which comprises a variable domain (VL) and a constant domain (CL),

[0156] (ii) a first heavy chain comprising a variable domain (VH), a first constant domain (CH1), optionally a hinge, a second constant domain (CH2) and a third constant domain (CH3), said CH2 and CH3 domains forming a first Fc chain,

[0157] wherein the first light chain and the heavy chain form a first binding moiety that binds to a target specifically expressed on the surface of a T cell, and

[0158] (iii) a second light chain, which comprises a variable domain (VL) and a constant domain (CL),

[0159] (iv) a second heavy chain comprising a variable domain (VH), a first constant domain (CH1), optionally a hinge, a second constant domain (CH2) and a third constant domain (CH3), said CH2 and CH3 domains forming a second Fc chain,

[0160] wherein the second light chain and the heavy chain form a second binding moiety that binds to CD28, and

[0161] The first and second Fc chains are complementary and form an Fc domain.

[0162] In an embodiment, the first binding moiety is a Fab and the second binding moiety is a CrossMAb, such that the multifunctional molecule preferably comprises or consists of:

[0163] (i) a first chain, which is a light chain comprising a variable domain (VL) and a constant domain (CL),

[0164] (i) a second chain, which is a heavy chain comprising a variable domain (VH) and a constant domain (CH1),

[0165] The light chain and the heavy chain form a first binding moiety that binds to a target specifically expressed on the surface of a T cell;

[0166] (iii) a third chain, which comprises a variable domain (VH) and a constant domain (CL),

[0167] (iv) a fourth chain, which comprises a variable domain (VL) and a constant domain (CH1),

[0168] The first and second chains form a second binding moiety that binds to CD28.

[0169] Such molecules may further comprise an Fc domain, such that the multifunctional molecule preferably comprises two different Fab domains and an Fc domain. In particular, the multifunctional molecule comprises or consists of:

[0170] (i) a first chain, which is a light chain comprising a variable domain (VL) and a constant domain (CL),

[0171] (ii) a second chain, which is a heavy chain comprising a variable domain (VH), a first constant domain (CH1), optionally a hinge, a second constant domain (CH2) and a third constant domain (CH3); the CH2 and CH3 domains form a first Fc chain,

[0172] wherein the first and second chains form a first binding moiety that binds to a target specifically expressed on the surface of an immune cell,

[0173] (iii) a third chain comprising a variable domain (VH), a first constant domain (CL), optionally a hinge, a second constant domain (CH2), and a third constant domain (CH3); the CH2 and CH3 domains form a second Fc chain; and

[0174] (iv) a fourth chain, which comprises a variable domain (VL) and a constant domain (CH1),

[0175] wherein the third and fourth chains form a second binding moiety that binds to CD28, and

[0176] Wherein the first and second Fc chains are complementary and form an Fc domain.

[0177] In another embodiment, the first binding moiety is a Fab and the second binding moiety is a scFv, such that the multifunctional molecule preferably comprises or consists of:

[0178] (i) a first light chain, which comprises a variable domain (VL) and a constant domain (CL),

[0179] (ii) a first heavy chain, which comprises a variable domain (VH) and a constant domain (CH1),

[0180] wherein the first light chain and the heavy chain form a first binding portion that binds to a target specifically expressed on the surface of a T cell and forms a Fab,

[0181] (iii) a second light chain, which comprises a variable domain (VL), and

[0182] (iv) a second heavy chain comprising a variable domain (VH),

[0183] The second light chain and the heavy chain are connected via a peptide linker to form a scFv that binds to CD28.

[0184] Such molecules may further comprise an Fc domain, such that the multifunctional molecule comprises a Fab, a scFv and an Fc domain. In particular, the multifunctional comprises or consists of:

[0185] (i) a first light chain, which comprises a variable domain (VL) and a constant domain (CL),

[0186] (ii) a first heavy chain comprising a variable domain (VH), a first constant domain (CH1), optionally a hinge, a second constant domain (CH2) and a third constant domain (CH3), said CH2 and CH3 domains forming a first Fc chain,

[0187] wherein the first light chain and the heavy chain form a first binding portion that binds to a target specifically expressed on the surface of a T cell and forms a Fab,

[0188] (iii) a second light chain, comprising a variable domain (VL),

[0189] (iv) a second heavy chain comprising a variable domain (VH),

[0190] wherein the second light chain and the heavy chain are connected via a peptide linker to form a scFv that binds to CD28, and

[0191] (v) a second Fc chain, which is complementary to the first Fc chain to form an Fc domain.

[0192] In particular, the multifunctional molecule of the present invention comprises or consists of:

[0193] (i) a first chain comprising or essentially consisting of a light chain variable domain (VL) and a light chain constant domain (CL),

[0194] (ii) a second chain comprising or essentially consisting of a heavy chain variable domain (VH), a first heavy chain constant domain (CH1), optionally a hinge, a second heavy chain constant domain (CH2) and a third heavy chain constant domain (CH3), said CH2 and CH3 domains forming a first Fc chain,

[0195] wherein the first and second chains form a first binding moiety that binds to a target specifically expressed on the surface of a T cell (e.g., PD-1) and form a Fab;

[0196] (iii) a third chain comprising or consisting essentially of: a) a variable domain (VL) connected to a variable domain (VH), preferably via a peptide linker, forming a second binding moiety, which is a scFV that binds to CD28; and b) optionally a hinge and a second Fc chain, complementary to the first Fc chain to form an Fc domain.

[0197] The third chain forms the second binding part that binds to CD28.

[0198] In an embodiment, the first binding moiety is CrossMAb and the second binding moiety is ScFV, such that the multifunctional molecule preferably comprises or consists of:

[0199] (i) a first chain comprising a light chain variable domain (VL) and a heavy chain constant domain (CH1),

[0200] (i) a second chain comprising a heavy chain variable domain (VH) and a light chain constant domain (CL),

[0201] The first and second chains form a CrossMAb that binds to a target specifically expressed on the surface of T cells (preferably PD-1);

[0202] (iii) a third chain comprising a variable domain (VH) and a variable domain (VL), preferably connected by a peptide linker, said third chain forming a scFv that binds to CD28.

[0203] Such molecules may further comprise an Fc domain. In particular, the multifunctional molecule comprises or consists of:

[0204] (i) a first chain comprising a heavy chain variable domain (VH) and a light chain constant domain (CL),

[0205] (ii) a second chain comprising a light chain variable domain (VL) and a heavy chain constant domain (CH1), a second constant domain (CH2) and a third constant domain (CH3); the CH2 and CH3 domains form a first Fc chain,

[0206] wherein the first and second chains form a first binding moiety that binds to a target specifically expressed on the surface of an immune cell (e.g., PD-1);

[0207] (iii) a third chain comprising a) a variable domain (VL) connected to a variable domain (VH), preferably via a peptide linker, to form a second binding moiety, which is a scFV that binds to CD28, and b) a second Fc chain, which is complementary to the first Fc chain to form an Fc domain.

[0208] In particular, the scFv may be linked to the N-terminus of the second Fc chain, to the C-terminus of the first or second Fc chain or to the C-terminus of the CL of the first binding moiety, preferably to the N-terminus of the second Fc chain or to the C-terminus of the first Fc chain, even more preferably to the N-terminus of the second Fc chain.

[0209] In certain embodiments, the second Fc chain does not contain any binding moiety, immunotherapeutic agent or other protein.

[0210] Alternatively, the second Fc chain may be linked at its C-terminus to another binding moiety or immunotherapeutic agent or protein that is not the moiety that binds CD28.

[0211] In another embodiment, the first binding moiety is F(ab')2 and the second binding moiety is scFv. In particular, the multifunctional molecule comprises or consists of:

[0212] (i) a first light chain, which comprises a variable domain (VL) and a constant domain (CL),

[0213] (ii) a first heavy chain, which comprises a variable domain (VH) and a constant domain (CH1),

[0214] (iii) a second light chain, which comprises a variable domain (VL) and a constant domain (CL), and

[0215] (iv) a second heavy chain comprising a variable domain (VH) and a constant domain (CH1),

[0216] wherein the first and second light chains and the second light chain and heavy chain form a first binding portion F(ab')2 that binds to a target specifically expressed on the surface of a T cell,

[0217] (v) a third light chain, comprising a variable domain (VL),

[0218] (iv) a third heavy chain, comprising a variable domain (VH),

[0219] The third light chain and the heavy chain are connected via a peptide linker to form a second binding part (scFv) that binds to CD28.

[0220] In a specific embodiment, F(ab')2 comprises Fab and CrossMab, such that the multifunctional molecule comprises or consists of:

[0221] (i) a first light chain, which comprises a variable domain (VL) and a constant domain (CH1),

[0222] (ii) a first heavy chain, which comprises a variable domain (VH) and a constant domain (CL),

[0223] (iii) a second light chain, which comprises a variable domain (VL) and a constant domain (CL),

[0224] (iv) a second heavy chain comprising a variable domain (VH) and a constant domain (CH1),

[0225] wherein the first and second light chains and the first and second heavy chains form a first binding moiety that binds to a target specifically expressed on the surface of a T cell,

[0226] (v) a third light chain, comprising a variable domain (VL),

[0227] (iv) a third heavy chain, comprising a variable domain (VH),

[0228] The third light chain and the heavy chain are connected via a peptide linker to form a second binding part (scFv) that binds to CD28.

[0229] In particular, the multifunctional molecule comprises or consists of:

[0230] (i) a first chain comprising or essentially consisting of a light chain variable domain (VL) and a light chain constant domain (CL),

[0231] (ii) a second chain comprising or essentially consisting of a heavy chain variable domain (VH) and a heavy chain constant domain (CH1),

[0232] (iii) a third chain comprising or essentially consisting of a light chain variable domain (VL) and a light chain constant domain (CL),

[0233] (iv) a fourth chain comprising or essentially consisting of a heavy chain variable domain (VH) and a heavy chain constant domain (CH1),

[0234] wherein the first, second, third and fourth chains form a first F(ab')2 binding portion that binds to a target specifically expressed on the surface of a T cell (e.g., PD-1); and

[0235] (v) a fifth chain comprising or essentially consisting of a variable domain (VL) connected to a variable domain (VH), preferably via a peptide linker, forming a second binding moiety which is a scFV that binds to CD28.

[0236] Such molecules may further comprise an Fc domain, such that the multifunctional molecule comprises F(ab')2, scFv and an Fc domain. In particular, the multifunctional molecule comprises or consists of:

[0237] (i) a first light chain, which comprises a variable domain (VL) and a constant domain (CL),

[0238] (ii) a first heavy chain comprising a variable domain (VH) and a constant domain (CH1), optionally a hinge, a second constant domain (CH2) and a third constant domain (CH3); said CH2 and CH3 domains forming a first Fc chain,

[0239] (iii) a second light chain, which comprises a variable domain (VL) and a constant domain (CL),

[0240] (iv) a second heavy chain comprising a variable domain (VH) and a constant domain (CH1), optionally a hinge, a second constant domain (CH2) and a third constant domain (CH3); said CH2 and CH3 domains forming a second Fc chain,

[0241] wherein the first and second Fc chains are complementary and form an Fc domain, and wherein the first and second light chains and the first and second heavy chains form a first binding moiety (antibody) that binds to a target specifically expressed on the surface of a T cell,

[0242] (v) a third light chain, comprising a variable domain (VL),

[0243] (iv) a third heavy chain, comprising a variable domain (VH),

[0244] The third light chain and the heavy chain are connected via a peptide linker to form a second binding part (scFv) that binds to CD28.

[0245] In particular, the multifunctional molecule comprises or consists of:

[0246] (i) a first chain comprising or essentially consisting of a light chain variable domain (VL) and a light chain constant domain (CL),

[0247] (ii) a second chain comprising or essentially consisting of a heavy chain variable domain (VH) and a heavy chain constant domain (CH1), optionally a hinge, a second heavy chain constant domain (CH2) and a third heavy chain constant domain (CH3); said CH2 and CH3 domains forming a first Fc chain,

[0248] (iii) a third chain comprising or essentially consisting of a light chain variable domain (VL) and a light chain constant domain (CL),

[0249] (iv) a fourth chain comprising or essentially consisting of a heavy chain variable domain (VH) and a first heavy chain constant domain (CH1), optionally a hinge, a second heavy chain constant domain (CH2) and a third heavy chain constant domain (CH3); said CH2 and CH3 domains forming a second Fc chain,

[0250] wherein the first and second Fc chains are complementary and form an Fc domain, and wherein the first, second, third, and fourth chains form a first binding moiety, which is an antibody that binds to a target specifically expressed on the surface of a T cell (such as PD-1); and

[0251] (vi) a fifth chain comprising or essentially consisting of a variable domain (VL) connected to a variable domain (VH), preferably via a peptide linker, forming a second binding moiety which is a scFV that binds to CD28.

[0252] In an embodiment, the molecule comprises F(ab')2, scFv and Fc domains containing Fab and CrossMAb. In particular, the multifunctional molecule comprises or consists of:

[0253] (i) a first light chain, which comprises a variable domain (VL) and a constant domain (CH1),

[0254] (ii) a first heavy chain comprising a variable domain (VH) and a constant domain (CL), optionally a hinge, a second constant domain (CH2) and a third constant domain (CH3); the CH2 and CH3 domains form a first Fc chain

[0255] (iii) a second light chain, which comprises a variable domain (VL) and a constant domain (CL),

[0256] (iv) a second heavy chain comprising a variable domain (VH) and a constant domain (CH1), optionally a hinge, a second constant domain (CH2) and a third constant domain (CH3), said CH2 and CH3 domains forming a first Fc chain;

[0257] wherein the first and second light chains and the first and second heavy chains form a first binding moiety (antibody) that binds to a target specifically expressed on the surface of a T cell

[0258] (v) a third light chain, comprising a variable domain (VL),

[0259] (iv) a third heavy chain, comprising a variable domain (VH),

[0260] The third light chain and the heavy chain are connected via a peptide linker to form a second binding part (scFv) that binds to CD28.

[0261] In particular, the scFv may be linked to the C-terminus of the first or second Fc chain, the C-terminus of the CL of the first or second light chain of the first binding moiety, or the N-terminus of the VH or VL of the first or second light chain of the first binding moiety.

[0262] In an embodiment, both the first and the second binding moiety are scFv, such that the multifunctional molecule preferably comprises or consists of:

[0263] (i) a first light chain, which comprises a variable domain (VL), and

[0264] (ii) a first heavy chain comprising a variable domain (VH),

[0265] The first light chain and the heavy chain are connected by a peptide linker and form a first binding portion that binds to a target specifically expressed on the surface of a T cell;

[0266] (iii) a second light chain, comprising a variable domain (VL),

[0267] (iv) a second heavy chain comprising a variable domain (VH),

[0268] The second light chain and heavy chain are connected by a peptide linker and form a second binding moiety that binds to CD28.

[0269] Such molecules may further comprise an Fc domain, such that the multifunctional molecule comprises or consists of:

[0270] (i) a first light chain, comprising a variable domain (VL),

[0271] (ii) a first heavy chain comprising a variable domain (VH), optionally a hinge, a CH2 and a CH3 domain; said CH2 and CH3 domains forming a first Fc chain,

[0272] wherein the first light chain and the heavy chain are connected by a peptide linker and form a first binding portion that binds to a target specifically expressed on the surface of a T cell; and,

[0273] (iii) a second light chain, which comprises a variable domain (VL) and a constant domain (CL),

[0274] (iv) a second heavy chain comprising a variable domain (VH), a first constant domain (CH1), optionally a hinge, a second constant domain (CH2) and a third constant domain (CH3); said CH2 and CH3 domains forming a second Fc chain,

[0275] wherein the second light chain and the heavy chain are connected via a peptide linker and form a second binding portion that binds to CD28, and,

[0276] The first and second Fc chains are complementary to form an Fc domain.

[0277] The different elements making up the multifunctional molecules of the present invention are described in more detail below.

[0278] Combination part

[0279] According to the present invention, the multifunctional molecule comprises:

[0280] (a) a first binding moiety that binds to a target specifically expressed on a T cell, particularly a T cell surface; and

[0281] (a) a second binding moiety which is an agonist of CD28 and is an anti-CD28 antigen binding domain;

[0282] The multifunctional molecule comprises a single anti-CD28 antigen binding domain.

[0283] As described above, the multifunctional molecule may in particular comprise two, three or four binding moieties that bind to a target specifically expressed on the surface of a T cell, but only one binding moiety binds to CD28. For example, the multifunctional molecule may comprise one, two or three binding moieties that bind to a target specifically expressed on the surface of a T cell, and one binding moiety that binds to CD28.

[0284] The binding moieties are described in more detail below. The "first binding moiety" moiety is an example of a binding moiety that can be used as "one, two or three binding moieties that bind to a target specifically expressed on the surface of a T cell", which can be included in a multifunctional molecule according to the invention. The "second binding moiety" moiety is an example of a binding moiety for CD28.

[0285] The first and second binding moieties are preferably antigen binding domains, particularly derived from Fab, Fab', F(ab')2, Fv, single chain (scFv), CrossMAb or nanobody (VHH), preferably F(ab')2, Fab, CrossMAb or scFV antigen binding domains. In the multifunctional molecule, the first and second binding moieties may have the same or different configurations. In a specific aspect, the first and second binding moieties are not both scFv. In a preferred aspect, the anti-CD28 antigen binding moiety is an scFv, and the binding moiety that binds to a target specifically expressed on the surface of an immune cell, particularly a T cell, is not an scFv.

[0286] In one embodiment, the first or second binding moiety is a CrossMab.

[0287] Preferably, the anti-CD28 antigen binding portion is a scFv, and the binding portion that binds to a target specifically expressed on the surface of an immune cell comprises or consists of F(ab')2, Fab or CrossMAb.

[0288] The first binding part

[0289] The multifunctional molecule according to the invention comprises a first binding moiety which binds to a target expressed on T cells, in particular T cells present in the tumor environment (e.g., T cells recruited to the tumor site), such as PD-1 positive T cells.

[0290] The target specifically expressed on the surface of T cells is selected from the group consisting of PD-1, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, TIM3, CD39, CD30, NKG2D, NKG2A, LAG3, 2B4, DR3, CD101, CD44, CD38, CXCR3, CXCR5, CD4, CD8, CD25, CRTAM, CD96, CD226, CD112R, CD103, CEACAM, and CD122. Preferably, the target specifically expressed on the surface of T cells is selected from the group consisting of PD-1, VISTA, CTLA-4, BTLA, TIGIT, CD160, LAG3, and TIM3, preferably PD-1. In particular, these markers are not antigens of the TCR pathway (interaction between antigen presenting cells and T cells).

[0291] Preferably, the target specifically expressed on the surface of T cells is a target that allows interaction between T cells and tumor cells (e.g., PD-1 on T cells and PD-L1 on tumor cells). In particular, the target specifically expressed on the surface of T cells is not a target that allows interaction between T cells and antigen presenting cells (APCs) via the TCR pathway.

[0292] As used herein, the term "target" of a binding moiety refers to a carbohydrate, lipid, peptide, polypeptide, protein, antigen or epitope that is specifically recognized or targeted by a first binding moiety according to the invention and expressed on the outer surface of a T cell. With regard to the expression of a target on the surface of a T cell, the term "expression" refers to a target that is present or presented on the outer surface of the cell, such as a carbohydrate, lipid, peptide, polypeptide, protein, antigen or epitope.

[0293] In one aspect, the target is specifically expressed by T cells in healthy subjects or subjects with diseases (particularly such as cancer). In one aspect, the target is specifically expressed by T cells in subjects with diseases such as cancer. Preferably, the target has a higher expression level in T cells in subjects with diseases such as cancer than in T cells in healthy subjects. This means that the target has a higher expression level in T cells than in other cells, or the ratio of T cells expressing the target to total immune cells is higher than the ratio of other cells expressing the target to total other cells. Preferably, the expression level or ratio is 2, 5, 10, 20, 50 or 100 times higher. More specifically, it can be directed to T cells, more specifically CD4+ T cells, CD8+ T cells, effector T cells or exhaustion T cells, or in a specific context, for example, patients with diseases such as cancer or infection.

[0294] As used herein, "T cells" or "T lymphocytes" include, for example, CD4+ T cells, CD8+ T cells, T helper type 1 T cells, T helper type 2 T cells, T regulatory cells, T helper type 17 T cells, and suppressor T cells. In very specific aspects, the T cells are exhausted T cells or effector memory stem cell-like T cells.

[0295] The target can be a receptor expressed on the surface of a T cell. The receptor can be an inhibitory receptor. Alternatively, the receptor can be an activating receptor.

[0296] Advantageously, the first binding moiety does not bind to CD28 or any variant or mutant thereof.Alternatively or additionally, the first binding moiety does not bind to the CD28 ligands, ie B7.1 and B7.2 or any variant or mutant thereof.

[0297] Such targets expressed on T cells are described in more detail in Table D below.

[0298] Table D: Examples of Targets of Interest

[0299]

[0300]

[0301]

[0302] In a specific aspect, the T cell is a depleted T cell or an effector memory stem cell-like T cell, and the target is a factor expressed on the surface of the depleted T cell or the effector memory stem cell-like T cell. T cell exhaustion is a state in which T cells gradually lose function, proliferation ability and cytotoxic potential, ultimately leading to their death. T cell exhaustion can be caused by a variety of factors, such as continuous antigen exposure or inhibitory receptors, including PD-1, TIM3, CTLA-4, LAG-3, BTLA, TIGIT and CD160. Preferably, this factor, particularly this exhaustion factor, is selected from the group consisting of PD-1, TIM3, CTLA-4, LAG3, BTLA, TIGIT and CD160.

[0303] In a preferred aspect, the first binding moiety is an antigen binding domain that has antagonist activity against a target, particularly a checkpoint inhibitor between tumor cells and T cells, such as PD-1.

[0304] As used herein, the term "antagonist" refers to a substance that blocks or reduces the activity or function of another substance. In particular, the term refers to a binding domain that binds to a cell receptor (e.g., PD-1) such as a reference substance (e.g., PD-L1 and / or PD-L2), preventing it from producing all or part of its usual biological effects (e.g., producing an immunosuppressive microenvironment). Antagonist activity can be assessed by competitive ELISA.

[0305] In an embodiment, the first binding moiety is an antigen binding domain, in particular an antigen binding domain derived from an antibody. This means in particular that when it comes to antibody fragments, such as F(ab')2, Fab, Fab' or scFV, such fragments, in particular the VL and VH chains, are from antibodies, in particular antibodies that have been described in the art.

[0306] In a preferred aspect, the first binding moiety of the multifunctional molecule is an antibody, fragment or derivative thereof specific for an immune checkpoint inhibitor.

[0307] Even more preferably, the multifunctional molecule of the present invention comprises a first binding moiety which binds to a target specifically expressed on the surface of T cells, the target being selected from the group consisting of PD-1, VISTA, CTLA-4, BTLA, TIGIT, CD160, LAG3 and TIM3.

[0308] In a preferred aspect, the first binding portion of the multifunctional molecule is an antibody, a fragment or derivative thereof, such as F(ab')2, Fab, Fab' or a single-chain variable fragment (scFV), which is specific for PD-1, VISTA, CTLA-4, BTLA, TIGIT, CD160, LAG3 and TIM3.

[0309] Preferably, the first binding moiety of the multifunctional molecule is a F(ab')2, Fab, Fab' or CrossMAb specific for PD-1, VISTA, CTLA-4, BTLA, TIGIT, CD160, LAG3 and TIM3 (preferably for PD-1). Even more preferably, the first binding moiety of the multifunctional molecule is a Fab or CrossMAb specific for PD-1, VISTA, CTLA-4, BTLA, TIGIT, CD160, LAG3 and TIM3 (preferably for PD-1).

[0310] In a preferred aspect, the first binding portion of the multifunctional molecule is from or derived from an antibody, a fragment or derivative thereof, such as F(ab')2, Fab, Fab' or a single chain variable fragment (scFV), which is specific for PD-1, VISTA, CTLA-4, BTLA, TIGIT, CD160, LAG3 and TIM3. This means that the first binding portion comprises at least an antigen binding domain (i.e., the CDR or VH and VL domains of the antibody).

[0311] Many antibodies against PD-1, TIM3, VISTA, CTLA-4, LAG-3, BTLA, TIGIT, and CD160 have been described in the art.

[0312] As used herein, the terms "programmed death 1", "programmed cell death 1", "PD1", "PD-1", "PDCD1", "PD-1 antigen", "human PD-1", "hPD-1" and "hPD1" are used interchangeably and refer to the programmed death-1 receptor, also known as CD279, and include variants and isoforms of human PD-1, and analogs that have at least one common epitope with PD-1. PD-1 is a key regulator of immune response thresholds and peripheral immune tolerance. It is expressed on activated T cells, B cells, monocytes, and dendritic cells, and binds to its ligands PD-L1 and PD-L2. Human PD-1 is encoded by the PDCD1 gene. For example, the amino acid sequence of human PD-1 has been published under GenBank accession number NP_005009. PD1 has four splice variants that are expressed on human peripheral blood mononuclear cells (PBMCs). Therefore, the PD-1 protein includes full-length PD-1, as well as alternative splice variants of PD-1, such as PD-1Aex2, PD-1Aex3, PD-1Aex2, 3, and PD-1Aex2, 3, 4. Unless otherwise indicated, these terms include any variants and isoforms of human PD-1 naturally expressed by PBMCs or expressed by cells transfected with the PD-1 gene.

[0313] There are a variety of anti-PD-1 antibodies that have been clinically approved, and some others are still in clinical development. For example, the anti-PD1 antibody can be selected from the following group: pembrolizumab (also known as Keytruda, lambrolizumab, MK-3475, SCH-900475), nivolumab (OPDIVO, MDX-1106, BMS-936558, ONO-4538), pidilizumab (CT-011), cemiplizumab (Libtayo), carrelizumab, AUNP12, AMP-224, AGEN-2034 (Balstilimab), BGB-A317 (tislelizumab), PDR001 (s partalizumab), MK-3477, PF-06801591 (Sasanlimab), JNJ-63723283 (Cetrelimab), genolimzumab (CBT-501), LZM-009 (Lipustobart), BCD-100 (Palolimab), SHR-1201, BAT-1306, AK-103 (HX-008), MEDI-0680 (also known as AMP-514), JS001 (see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), BI-754091, CBT-501, INCSHR1210 (also known as SHR-1210), TSR-042 (also known as ANB011), GLS-010 (also known as WBP3055), AM-0001 (Armo), STI-1110 (see WO 2014 / 194302), MGA012 (see WO 2017 / 19846), or IBI308 (see WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825, and WO 2017 / 133540, the disclosures of which are incorporated herein by reference), described in WO 2006 / 121168, the disclosure of which is incorporated herein by reference. Multifunctional molecules targeting PD-1 are also known as RG7769 (Roche), XmAb20717 (Xencor), MEDI5752 (AstraZeneca), FS118 (F-star), SL-279252 (Takeda), and XmAb23104 (Xencor).

[0314] In some aspects, the target is PD-1 and the first binding moiety is specific for PD-1. Preferably, the first binding moiety is an antagonist of PD-1. Even more preferably, the anti-PD-1 antibody is pembrolizumab, nivolumab or OSE-279 (as described in WO2020 / 127366, the disclosure of which is incorporated herein by reference).

[0315] In specific aspects, the anti-PD1 antibody can be pembrolizumab (also known as Keytrudalambrolizumab, MK-3475) or nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538).

[0316] Antibodies against TIM3 and multifunctional molecules targeting TIM3 are also referred to as Sym023, TSR-022, MBG453, LY3321367, INCAGN02390, BGTB-A425, LY3321367, RG7769 (Roche). In some aspects, TFM-3 antibodies are disclosed in International Patent Application Publication Nos. WO2013006490, WO2016 / 161270, WO 2018 / 085469 or WO 2018 / 129553, WO 2011 / 155607, US 8,552,156, EP 2581113 and US 2014 / 044728, the disclosures of which are incorporated herein by reference.

[0317] Antibodies against CTLA-4 and multifunctional molecules targeting CTLA-4 are also known as ipilimumab, tremelimumab, MK-1308, AGEN-1884, XmAb20717 (Xencor), MEDI5752 (AstraZeneca). Anti-CTLA-4 antibodies are also disclosed in WO18025178, WO19179388, WO19179391, WO19174603, WO19148444, WO19120232, WO19056281, WO19023482, WO18209701, WO18165895, WO18160536, WO18156250, WO18106862, WO18106863, WO18106864, WO18106865, WO18106866, WO18106867, WO18106868 6864, WO18068182, WO18035710, WO18025178, WO17194265, WO17106372, WO17084078, WO17087588, WO16196237, WO16130898, WO16015675, WO12120125, WO09100140 and WO07008463, the disclosures of which are incorporated herein by reference.

[0318] Antibodies against LAG3 and multifunctional molecules targeting LAG-3 are also referred to as BMS-986016, IMP701, MGD012 or MGD013 (bispecific PD-1 and LAG-3 antibodies). Anti-LAG-3 antibodies are also disclosed in WO2008132601, EP2320940 and WO19152574, the disclosures of which are incorporated herein by reference.

[0319] Antibodies against BTLA are also referred to in the art as hu Mab8D5, hu Mab8A3, hu Mab21H6, huMab19A7 or hu Mab4C7. Antibody TAB004 against BTLA is currently undergoing clinical trials in patients with advanced malignancies. Anti-BTLA antibodies are also disclosed in WO08076560, WO10106051 (such as BTLA8.2), WO11014438 (such as 4C7), WO17096017 and WO17144668 (such as 629.3), the disclosures of which are incorporated herein by reference.

[0320] Antibodies against TIGIT are also known in the art as BMS-986207 or AB154, BMS-986207 disclosed in WO19232484, CPA.9.086, CHA.9.547.18, CPA.9.018, CPA.9.027, CPA.9.049, CPA.9.057, CPA.9.059, CPA.9.083, CPA.9.089, CPA.9.093, CPA.9.101, CPA.9.103, C HA.9.536.1, CHA.9.536.3, CHA.9.536.4, CHA.9.536.5, CHA.9.536.6, CHA.9.536.7, CHA.9.536.8, CHA.9.560.1, CHA.9.560.3, CHA.9.560.4, CHA.9 .560.5, CHA.9.560.6, CHA.9.560.7, CHA.9.560.8, CHA.9.546.1, CHA.9.547.1, CHA.9.547.2, CHA.9.547.3, CHA.9.547.4, CHA.9.547.6, CHA.9.547 .7, CHA.9.547.8, CHA.9.547.9, CHA.9.547.13, CHA.9.541.1, CHA.9.541.3, CHA.9.541.4, CHA.9.541.5, CHA.9.541.6, CHA.9.541.7 and CHA.9.541.8. Anti-TIGIT antibodies are also disclosed in WO16028656, WO16106302, WO16191643, WO17030823, WO17037707, WO17053748, WO17152088, WO18033798, WO18102536, WO18102746, WO18160704, WO18200430, WO18204363, WO19023504, WO19062832, WO19129221, WO19129261, WO19137548, WO19152574, WO19154415, WO19168382 and WO19215728, the disclosures of which are incorporated herein by reference.

[0321] Antibodies against CD160 are also known in the art as CL1-R2 CNCM I-3204 as disclosed in WO06015886 or other antibodies disclosed in WO10006071, WO10084158, WO18077926, the disclosures of which are incorporated herein by reference.

[0322] In another specific aspect, the target is PD-1, and the first binding part of the multifunctional molecule is an antigen binding domain, in particular an antibody, a fragment or a derivative thereof, such as a Fab or scFv or an antibody mimetic, that is specific for PD-1. Then, in a specific aspect, the antigen binding domain contained in the multifunctional molecule according to the present invention is an anti-PD1 antibody or an antigen binding fragment thereof, preferably a human, humanized or chimeric anti-PD1 antibody or an antigen binding fragment thereof.

[0323] In an embodiment, the target is PD-1, and the first binding moiety is a F(ab')2, Fab or scFv specific for PD-1, preferably a human, humanized or chimeric anti-PD1 Fab or scFv.

[0324] Preferably, the antigen binding domain is an antagonist of PD-1.

[0325] In a very specific aspect of the present disclosure, the first binding moiety is an antigen binding domain targeting PD-1 and is derived from an antibody disclosed in WO2020 / 127366, the disclosure of which is incorporated herein by reference.

[0326] Then, in an embodiment, the first binding moiety comprises an anti-PD-1 antigen binding domain comprising:

[0327] (i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and

[0328] (ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3,

[0329] in:

[0330] - a heavy chain CDR1 (HCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 1, optionally, in particular having one, two or three modifications at any position other than position 3 of SEQ ID NO: 1, selected from substitutions, additions, deletions and any combination thereof;

[0331] - a heavy chain CDR2 (HCDR2) comprising or consisting of the amino acid sequence of SEQ ID NO: 2, optionally having one, two or three modifications, in particular at any position other than positions 13, 14 and 16 of SEQ ID NO: 2, selected from substitutions, additions, deletions and any combination thereof;

[0332] - a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 3, optionally having one, two or three modifications, in particular at any position other than positions 2, 3, 7 and 8 of SEQ ID NO: 3, selected from substitutions, additions, deletions and any combination thereof;

[0333] - the light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 4, optionally, in particular has one, two or three modifications at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID NO: 4, selected from substitutions, additions, deletions and any combination thereof;

[0334] - the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 5, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; and

[0335] - The light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 6, optionally having one, two or three modifications at any position other than positions 1, 4 and 6 of SEQ ID NO: 6, the modifications being selected from substitution, addition, deletion and any combination thereof.

[0336] In another aspect, the anti-PD-1 antigen binding domain comprises or consists essentially of: (i) a heavy chain variable region (VH) comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3; and (ii) a light chain variable region (VL) comprising CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 6.

[0337] Preferably, the CDRs of such anti-PD1 binding domain have been determined by the Kabat method.

[0338] In one aspect, the anti-PD-1 antigen binding domain comprises a framework region, in particular a heavy chain variable region framework region (HFR) HFR1, HFR2, HFR3 and HFR4 and a light chain variable region framework region (LFR) LFR1, LFR2, LFR3 and LFR4, in particular HFR1, HFR2, HFR3 and HFR4, optionally, in particular HFR3, i.e., any position other than positions 27, 29 and 32 of SEQ ID NO: 9 has one, two or three modifications selected from substitutions, additions, deletions and any combination thereof. Preferably, the anti-PD-1 antigen binding domain comprises HFR1 of SEQ ID NO: 7, HFR2 of SEQ ID NO: 8, HFR3 of SEQ ID NO: 9 and HFR4 of SEQ ID NO: 10. In addition, the anti-PD-1 antigen binding domain may comprise light chain variable region framework regions (LFRs) LFR1, LFR2, LFR3 and LFR4, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof. Preferably, the anti-PD-1 antigen binding domain comprises LFR1 of SEQ ID NO: 11, LFR2 of SEQ ID NO: 12, LFR3 of SEQ ID NO: 13 and LFR4 of SEQ ID NO: 14.

[0339] In one aspect, the anti-PD1 antigen binding domain comprises or consists essentially of:

[0340] (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 15, optionally, in particular having one, two or three modifications at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID NO: 15, the modifications being selected from substitutions, additions, deletions and any combination thereof;

[0341] (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 16, optionally, in particular having one, two or three modifications at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID NO: 16, the modifications being selected from substitutions, additions, deletions and any combination thereof.

[0342] Preferably, one, two or three modifications are outside the CDRs (ie, within the framework regions).

[0343] In one aspect, the anti-PD1 antigen binding domain comprises or consists essentially of:

[0344] (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 15, or a sequence having 80%, 85%, 90%, 95%, 97% or 99% sequence identity thereto; and

[0345] (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 16, or a sequence having 80%, 85%, 90%, 95%, 97% or 99% sequence identity thereto.

[0346] In another aspect, the anti-PD1 antigen binding domain comprises or consists essentially of:

[0347] (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 15; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 16.

[0348] In another embodiment, the first binding moiety comprises an anti-PD-1 antigen binding domain comprising:

[0349] (i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and

[0350] (ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3,

[0351] in:

[0352] - heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 65;

[0353] - heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 66;

[0354] - heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 67;

[0355] - light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 68;

[0356] - the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 69, and

[0357] - the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 70.

[0358] Preferably, the anti-PD-1 antigen binding domain comprises i) a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 71; and ii) a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 72.

[0359] In another embodiment, the first binding moiety comprises an anti-PD-1 antigen binding domain comprising:

[0360] (i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and

[0361] (ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3,

[0362] in:

[0363] - heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 73;

[0364] - heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 74;

[0365] - heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 75;

[0366] - light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 76;

[0367] - the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 77, and

[0368] - the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 78.

[0369] Preferably, the anti-PD-1 antigen binding domain comprises i) a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 79; and ii) a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 80.

[0370] In an embodiment, the anti-PD-1 antigen binding domain comprises VH, VL, CH1, and CL domains, such that the antigen binding domain is a Fab.

[0371] In such embodiments, the heavy chain constant domain (CH1) comprises or consists essentially of the amino acid sequence of SEQ ID NO: 17; optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0372] Preferably, the light chain constant domain (CL) comprises or essentially consists of the amino acid sequence of SEQ ID NO: 18, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0373] In an embodiment, the anti-PD-1 antigen binding domain is Fab or Fab', Fab or F(ab')2, and comprises i) a VH domain and a CH1 domain, the VH and CH1 domains having the amino acid sequences as shown in SEQ ID NOs: 15 and 16, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; and ii) a VL domain and a CL domain, the domains having the amino acid sequences as shown in SEQ ID NOs: 16 and 18, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0374] Preferably, the first binding moiety is an anti-PD-1 Fab or F(ab')2 comprising or consisting of: i) a chain comprising or consisting of a VH domain and a CH1 domain, the VH and CH1 domains having the amino acid sequences shown in SEQ ID NOs: 15 and 16, respectively, and ii) a chain comprising or consisting of a VL and CL domain, the domains having the amino acid sequences shown in SEQ ID NOs: 16 and 18, respectively.

[0375] In an embodiment, the first binding moiety is an anti-PD-1 Fab or F(ab')2 comprising or consisting of: i) a chain comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 19, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; and ii) a chain comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 20, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0376] In an embodiment, the first binding moiety comprises an anti-PD1 CrossMAb comprising or consisting of: i) a chain comprising or consisting of a VH and CL domain, said domains having an amino acid sequence as shown in SEQ ID NOs: 15 and 18, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; ii) a chain comprising or consisting of a VL and CH1 domain, said domains having an amino acid sequence as shown in SEQ ID NOs: 16 and 17, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0377] Preferably, the first binding moiety comprises an anti-PD1 CrossMAb comprising or consisting of: i) a chain comprising or consisting of a VH domain and a CL domain, said domains having the amino acid sequences shown in SEQ ID NOs: 15 and 18, respectively, and ii) a chain comprising or consisting of a VL and CH1 domain, said domains having the amino acid sequences shown in SEQ ID NOs: 16 and 17, respectively.

[0378] In an embodiment, the first binding moiety is an anti-PD-1 CrossMAb, which comprises or consists of: i) a chain comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 21, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; and ii) a chain comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 22, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0379] Preferably, one, two or three modifications in VH and / or VL or CH and / or CL are located outside the CDRs of any one of the above VH, VL, CH and / or CL (ie, within the framework regions).

[0380] The second binding part

[0381] The multifunctional molecule according to the present invention comprises a second binding moiety which is an agonist of CD28 and is an anti-CD28 antigen binding domain. The multifunctional molecule comprises a single second binding moiety such that the molecule comprises only one antigen binding domain that binds to CD28. In particular, the multifunctional molecule targeting CD28 is monovalent (only has a price for CD28).

[0382] As used herein, "CD28" refers to a co-stimulatory molecule expressed on the surface of various immune cells such as T cells. According to a specific embodiment, the CD28 protein refers to a human protein, as provided in the following GenBank numbers NP_001230006, NP_001230007, and NP_006130. Human CD28 cDNA was obtained free of charge from Dr. A. Aruffo and Dr. B. Seed, who published the sequence and the following partial document: Aruffo, A., and Seed, B., 1987, "Molecular cloning of a CD28 cDNA by a high efficiency COS cell expression system", Proc. Natl. Acad. Sci. USA, 84:8573. Therefore, the production of human CD28 cDNA can be seen in detail from this document. Several ligands for CD28 have been identified, B7.1 (also known as CD80) and B7.2 (also known as CD86). The B7.1 protein refers in particular to a human protein, as provided in the following GenBank Accession No. NP_005182. According to a specific embodiment, the B7.2 protein refers to a human protein, as provided in the following GenBank Accession No. NP_001193853.

[0383] The interaction of CD28 with its ligand triggers co-stimulatory signals that work synergistically with TCR signaling to promote T cell activation, proliferation, and function. CD28 signaling has been shown to modulate the threshold for T cell activation and to reduce the amount of TCR engagement required for T cell activation.

[0384] As used herein, the term "CD28 agonist" refers to an agonist that induces and / or increases CD28 biological function and / or expression. According to a specific embodiment, the CD28 agonist induces and / or increases CD28 signal transduction to immune cells (such as T cells); thereby inducing and / or increasing the immune co-stimulatory activity of CD28. In particular, the CD28 agonist promotes the immune response of effector T cells after the TCR activation signal.

[0385] According to specific embodiments, the CD28 agonist directly binds to CD28 and activates the CD28 receptor.

[0386] In certain embodiments, a CD28 agonist exhibits one or more desired functional properties, such as high affinity binding to CD28, e.g., binding to human CD28 at 10 7 M, 10 8 M, 10 9 M, 10 10M or less binding; lack of significant cross-reactivity with other immune checkpoint proteins, such as CTLA-4 and ICOS; ability to stimulate T cell proliferation; ability to increase IFN-γ and / or IL-2 secretion; ability to stimulate antigen-specific memory responses; ability to stimulate antibody responses and / or the ability to inhibit tumor cell growth.

[0387] In certain embodiments, the CD28 agonist is a Fab, Fab', single chain variable fragment (scFV) or single domain antibody (sdAb), preferably a Fab or scFV derived from an antibody, particularly a superagonist anti-CD28 antibody or a conventional anti-CD28 antibody.

[0388] In some cases, the anti-CD28 binding domain is not a sdAb or VHH.

[0389] As used herein, the term "conventional anti-CD28 antibody" refers to an antibody that binds CD28 (eg, a domain other than the basolateral domain) and co-stimulates T cells in a TCR-dependent mechanism.

[0390] As used herein, the term "superagonist anti-CD28 antibody" refers to an antibody that binds CD28 via the basolateral domain, resulting in polyclonal activation of T lymphocytes even in the absence of TCR stimulation.

[0391] Anti-CD28 antibodies suitable for use in the present invention can be generated using methods well known in the art. Alternatively, anti-CD28 antibodies recognized in the art can be used. Examples of anti-CD28 antibodies are disclosed in, for example, Poirier et al. (2012) American Journal of Transplantation 12(7): 1682-1690, Cell Immunol. 2005 Jul-Aug; 236(l-2): 154-60, which are incorporated herein by reference in their entirety.

[0392] Specific anti-CD28 antibodies that may be used according to some embodiments of the present invention include, but are not limited to, TAB08 (formerly known as TGN1412, produced by TheraMAB), a humanized monoclonal antibody against human CD28, an anti-CD28 agonistic antibody (humanized) provided by Bioscience catalog number: 100186, or a mouse monoclonal antibody against human CD28 (clone CD28.2, such as provided by eBioscience #16-0289-81).

[0393] Then, in embodiments, the second binding moiety comprises an anti-CD28 antigen binding domain comprising:

[0394] (i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and

[0395] (ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3,

[0396] in:

[0397] - a heavy chain CDR1 (HCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 30, optionally with one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof;

[0398] - a heavy chain CDR2 (HCDR2) comprising or consisting of the amino acid sequence of SEQ ID NO: 31, optionally with one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof;

[0399] - a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 32; optionally having one, two or three modifications selected from substitution, addition, deletion and any combination thereof;

[0400] - light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 33, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof;

[0401] - the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 34, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; and

[0402] - the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 35, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0403] In another aspect, the second antigen binding domain comprises or consists essentially of: (i) a heavy chain variable region (VH) comprising CDR1 of SEQ ID NO: 30, CDR2 of SEQ ID NO: 31, and CDR3 of SEQ ID NO: 32; and (ii) a light chain variable region (VL) comprising CDR1 of SEQ ID NO: 33, CDR2 of SEQ ID NO: 34, and CDR3 of SEQ ID NO: 35.

[0404] Preferably, the CDRs of such anti-CD28 binding domains have been determined by the Kabat method.

[0405] In one aspect, such anti-CD28 antigen binding domains comprise framework regions, in particular heavy chain variable region framework regions (HFR) HFR1, HFR2, HFR3 and HFR4 and light chain variable region framework regions (LFR) LFR1, LFR2, LFR3 and LFR4. Preferably, the anti-CD28 antigen binding domain comprises HFR1 of SEQ ID NO: 36, HFR2 of SEQ ID NO: 37, HFR3 of SEQ ID NO: 38 and HFR4 of SEQ ID NO: 39, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof. In addition, the anti-CD28 antigen binding domain may comprise LFR1 of SEQ ID NO: 40, LFR2 of SEQ ID NO: 41, LFR3 of SEQ ID NO: 42 and LFR4 of SEQ ID NO: 43, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof.

[0406] The framework regions and complementarity determining regions are preferably operably linked in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (from amino terminus to carboxyl terminus). In another embodiment, the second binding moiety comprises an anti-CD28 antigen binding domain comprising:

[0407] (i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and

[0408] (ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3,

[0409] in:

[0410] - a heavy chain CDR1 (HCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 91, optionally with one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof;

[0411] - a heavy chain CDR2 (HCDR2) comprising or consisting of the amino acid sequence of SEQ ID NO: 92, optionally with one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof;

[0412] - a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 93; optionally having one, two or three modifications selected from substitution, addition, deletion and any combination thereof;

[0413] - the light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 94, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof;

[0414] - the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 95, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; and

[0415] - the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 96, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0416] In another aspect, the second antigen binding domain comprises or consists essentially of: (i) a heavy chain variable region (VH) comprising CDR1 of SEQ ID NO: 91, CDR2 of SEQ ID NO: 92, and CDR3 of SEQ ID NO: 93; and (ii) a light chain variable region (VL) comprising CDR1 of SEQ ID NO: 94, CDR2 of SEQ ID NO: 96, and CDR3 of SEQ ID NO: 97.

[0417] Preferably, the CDRs of such anti-CD28 binding domains have been determined by the IMGT method.

[0418] In one aspect, the anti-CD28 antigen binding domain comprises a framework region, in particular a heavy chain variable region framework region (HFR) HFR1, HFR2, HFR3 and HFR4 and a light chain variable region framework region (LFR) LFR1, LFR2, LFR3 and LFR4. Preferably, the anti-CD28 antigen binding domain comprises HFR1 of SEQ ID NO: 97, HFR2 of SEQ ID NO: 98, HFR3 of SEQ ID NO: 99 and HFR4 of SEQ ID NO: 100, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof. In addition, the anti-CD28 antigen binding domain may comprise LFR1 of SEQ ID NO: 101, LFR2 of SEQ ID NO: 102, LFR3 of SEQ ID NO: 103 and LFR4 of SEQ ID NO: 104, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof.

[0419] These framework regions and complementarity determining regions are preferably operably linked in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (from amino terminus to carboxyl terminus).

[0420] In one aspect, the anti-CD28 antigen binding domain comprises or consists essentially of:

[0421] (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 44, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof;

[0422] (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 45, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof.

[0423] In another aspect, the anti-CD28 antigen binding domain comprises or consists essentially of:

[0424] (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 44; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 45.

[0425] Preferably, the anti-CD28 antigen binding domain is a scFV comprising or essentially consisting of a heavy chain variable region (VH) of SEQ ID NO: 44 and a light chain variable region (VL) of SEQ ID NO: 5, optionally connected by a peptide linker.

[0426] In an embodiment, the anti-CD28 antigen binding domain comprises VH, VL, CH1, and CL domains, such that the antigen binding domain is a Fab.

[0427] In such embodiments, the heavy chain constant domain (CH1) preferably comprises or essentially consists of SEQ ID NO: 46, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0428] Preferably, the light chain constant domain (CL) comprises or essentially consists of the amino acid sequence of SEQ ID NO: 47, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0429] In an embodiment, the anti-CD28 binding portion is a Fab comprising or consisting of: i) a chain comprising or consisting of a VH domain and a CH1 domain, said domains having the amino acid sequences as shown in SEQ ID NOs: 44 and 46, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; ii) a light chain comprising or consisting of a VL and CL domain, said domains having the amino acid sequences as shown in SEQ ID NOs: 45 and 47, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0430] In an embodiment, the anti-CD28 binding portion is a Fab comprising or consisting of: i) a chain comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 48, optionally with one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof, and ii) a chain comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 49, optionally with one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof.

[0431] In an embodiment, the anti-CD28 binding portion is a CrossMAb comprising or consisting of: i) a chain comprising or consisting of a VH domain and a CL domain, said domains having the amino acid sequences as shown in SEQ ID NOs: 44 and 47, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; ii) a light chain comprising or consisting of a VL and a CH1 domain, said domains having the amino acid sequences as shown in SEQ ID NOs: 45 and 46, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0432] Preferably, the anti-CD28 binding portion comprises a CrossMAb comprising or consisting of: i) a chain comprising or consisting of a VH domain and a CL domain having the amino acid sequences shown in SEQ ID NOs: 44 and 47, respectively, and ii) a light chain comprising or consisting of a VL and CH1 domain having the amino acid sequences shown in SEQ ID NOs: 45 and 46, respectively.

[0433] In an embodiment, the anti-CD28 binding portion is a Fab comprising or consisting of: i) a chain comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 50, optionally with one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof, and ii) a chain comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 51, optionally with one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof.

[0434] Preferably, one, two or three modifications in VH and / or VL or CH and / or CL are located outside the CDRs of any one of the above VH, VL, CH and / or CL (ie, within the framework regions).

[0435] In some embodiments, the anti-CD28 binding domain is a variant comprising one or more mutations in the CDR and / or framework region compared to the reference sequence. In particular, the variant is a functional conservative variant. In the context of the present invention, this means that the variant i) binds to CD28, ii) has agonist activity to CD28, and iii) when included in the multifunctional molecule of the present invention, can confer the characteristics of cis-activity to the multifunctional molecule. For example, a method for selecting a suitable multifunctional molecule comprising such a variant is disclosed in the "Method for Selecting a Suitable Multifunctional Molecules" section provided below.

[0436] In particular, the anti-CD28 binding domain comprises or consists essentially of:

[0437] (i) a heavy chain variable region (VH), comprising:

[0438] HFR1 of SEQ ID NO: 36; optionally having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 modifications selected from substitutions, additions, deletions and any combination thereof, preferably amino acid substitutions;

[0439] HCDR1 of SEQ ID NO: 30, optionally having 1, 2 or 3 modifications selected from substitution, addition, deletion and any combination thereof, preferably amino acid substitution;

[0440] HFR2 of SEQ ID NO: 37, optionally having 1, 2 or 3 modifications selected from substitution, addition, deletion and any combination thereof, preferably amino acid substitution;

[0441] HCDR2 of SEQ ID NO: 31, optionally having 1, 2 or 3 modifications selected from substitution, addition, deletion and any combination thereof, preferably amino acid substitution;

[0442] HFR3 of SEQ ID NO: 38; optionally having 1, 2, 3, 4, 5, 6 or 7 modifications selected from substitutions, additions, deletions and any combination thereof, preferably amino acid substitutions;

[0443] HCDR3 of SEQ ID NO: 32; optionally having 1, 2 or 3 modifications selected from substitution, addition, deletion and any combination thereof, preferably amino acid substitution; and

[0444] HFR4 of SEQ ID NO: 39, optionally having 1, 2 or 3 modifications selected from substitution, addition, deletion and any combination thereof, preferably amino acid substitution;

[0445] and (ii) a light chain variable region (VL) comprising:

[0446] LFR1 of SEQ ID NO: 40; optionally having 1, 2, 3, 4, 5, 6 or 7 modifications selected from substitutions, additions, deletions and any combination thereof, preferably amino acid substitutions;

[0447] LCDR1 of SEQ ID NO: 33, optionally having 1, 2 or 3 modifications selected from substitution, addition, deletion and any combination thereof, preferably amino acid substitution;

[0448] LFR2 of SEQ ID NO: 41, optionally having 1, 2 or 3 modifications selected from substitution, addition, deletion and any combination thereof, preferably amino acid substitution;

[0449] LCDR2 of SEQ ID NO: 34, optionally having 1, 2 or 3 modifications selected from substitution, addition, deletion and any combination thereof, preferably amino acid substitution;

[0450] LFR3 of SEQ ID NO: 42; optionally having 1, 2, 3, 4, 5, 6, 7 or 8 modifications selected from substitutions, additions, deletions and any combination thereof, preferably amino acid substitutions;

[0451] LCDR3 of SEQ ID NO: 35, optionally having 1, 2 or 3 modifications selected from substitution, addition, deletion and any combination thereof, preferably amino acid substitution; and

[0452] The LFR4 of SEQ ID NO: 43 optionally has 1, 2 or 3 modifications selected from substitution, addition, deletion and any combination thereof, preferably amino acid substitution.

[0453] Preferably, the anti-CD28 binding domain comprises or consists essentially of:

[0454] (i) a heavy chain variable region (VH), comprising:

[0455] HFR1 of SEQ ID NO: 36; optionally having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acid modifications selected from substitution, addition, deletion and any combination thereof, preferably amino acid substitution;

[0456] HCDR1 of SEQ ID NO: 30, optionally having 1 amino acid modification selected from substitution, addition, deletion, preferably amino acid substitution;

[0457] The HFR2 of SEQ ID NO: 37, optionally having 1 or 2 amino acid modifications selected from substitution, addition, deletion and any combination thereof, preferably amino acid substitution;

[0458] HCDR2 of SEQ ID NO: 31;

[0459] The HFR3 of SEQ ID NO: 38, optionally having 1, 2, 3, 4, 5, 6 or 7 amino acid modifications selected from substitution, addition, deletion and any combination thereof, preferably amino acid substitution;

[0460] HCDR3 of SEQ ID NO: 32; and

[0461] HFR4 of SEQ ID NO: 39, optionally having 1 amino acid modification selected from substitution, addition, deletion, preferably amino acid substitution;

[0462] and (ii) a light chain variable region (VL) comprising:

[0463] LFR1 of SEQ ID NO: 40; optionally having 1, 2, 3, 4, 5, 6 or 7 modifications selected from substitutions, additions, deletions and any combination thereof, preferably amino acid substitutions;

[0464] LCDR1 of SEQ ID NO: 33, optionally having 1 amino acid modification selected from substitution, addition, deletion, preferably amino acid substitution;

[0465] LFR2 of SEQ ID NO: 41, optionally having 1, 2 or 3 modifications selected from substitution, addition, deletion and any combination thereof, preferably amino acid substitution;

[0466] LCDR2 of SEQ ID NO: 34, optionally having 1 amino acid modification selected from substitution, addition, deletion, preferably amino acid substitution;

[0467] LFR3 of SEQ ID NO: 42; optionally having 1, 2, 3, 4, 5, 6, 7 or 8 modifications selected from substitutions, additions, deletions and any combination thereof, preferably amino acid substitutions;

[0468] LCDR3 of SEQ ID NO: 35; and

[0469] The LFR4 of SEQ ID NO: 43 optionally has 1 amino acid modification selected from substitution, addition, deletion, preferably amino acid substitution.

[0470] In another embodiment, the anti-CD28 binding domain comprises or consists essentially of:

[0471] (i) a heavy chain variable region (VH), comprising:

[0472] HFR1 of SEQ ID NO: 36; optionally having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acid modifications selected from substitution, addition, deletion and any combination thereof, preferably amino acid substitution;

[0473] HCDR1 of SEQ ID NO: 30, optionally having 1 amino acid modification selected from substitution, addition, deletion, preferably amino acid substitution;

[0474] The HFR2 of SEQ ID NO: 37, optionally having 1 or 2 amino acid modifications selected from substitution, addition, deletion and any combination thereof, preferably amino acid substitution;

[0475] HCDR2 of SEQ ID NO: 31;

[0476] The HFR3 of SEQ ID NO: 38, optionally having 1, 2, 3, 4, 5, 6 or 7 amino acid modifications selected from substitution, addition, deletion and any combination thereof, preferably amino acid substitution;

[0477] HCDR3 of SEQ ID NO: 32; and

[0478] HFR4 of SEQ ID NO: 39, optionally having 1 amino acid modification selected from substitution, addition, deletion, preferably amino acid substitution;

[0479] and (ii) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 45, optionally having 1, 2 or 3 modifications selected from substitutions, additions, deletions and any combination thereof, preferably amino acid substitutions; the modifications are preferably located in the framework regions.

[0480] In another embodiment, the anti-CD28 binding domain comprises or consists essentially of:

[0481] (i) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 44, optionally having 1, 2 or 3 modifications selected from substitutions, additions, deletions and any combination thereof, preferably amino acid substitutions; the modifications are preferably located in the framework regions; and

[0482] (ii) a light chain variable region (VL), comprising:

[0483] LFR1 of SEQ ID NO: 40; optionally having 1, 2, 3, 4, 5, 6 or 7 modifications selected from substitutions, additions, deletions and any combination thereof, preferably amino acid substitutions;

[0484] LCDR1 of SEQ ID NO: 33, optionally having 1 amino acid modification selected from substitution, addition, deletion, preferably amino acid substitution;

[0485] LFR2 of SEQ ID NO: 41, optionally having 1, 2 or 3 modifications selected from substitution, addition, deletion and any combination thereof, preferably amino acid substitution;

[0486] LCDR2 of SEQ ID NO: 34, optionally having 1 amino acid modification selected from substitution, addition, deletion, preferably amino acid substitution;

[0487] LFR3 of SEQ ID NO: 42; optionally having 1, 2, 3, 4, 5, 6, 7 or 8 modifications selected from substitutions, additions, deletions and any combination thereof, preferably amino acid substitutions;

[0488] LCDR3 of SEQ ID NO: 35; and

[0489] The LFR4 of SEQ ID NO: 43 optionally has 1 amino acid modification selected from substitution, addition, deletion, preferably amino acid substitution.

[0490] In another aspect, the anti-CD28 antigen binding domain comprises or consists essentially of:

[0491] (a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 44, 83, 84, 85 and 88, optionally having one, two or three amino acid modifications selected from substitution, addition, deletion and any combination thereof, preferably substitution; and (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 86, 87, 89 and 90, optionally having one, two or three amino acid modifications selected from substitution, addition, deletion and any combination thereof, preferably substitution.

[0492] Preferably, one, two or three modifications in VH and / or VL are outside the CDRs (ie, within the framework regions).

[0493] In another aspect, the anti-CD28 antigen binding domain comprises or consists essentially of:

[0494] (a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 44, 83, 84, 85 and 88; and (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 45, optionally having 1, 2 or 3 amino acid modifications selected from substitutions, additions, deletions and any combination thereof, preferably amino acid substitutions.

[0495] In another aspect, the anti-CD28 antigen binding domain comprises or consists essentially of:

[0496] (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 44, optionally having 1, 2 or 3 modifications selected from substitutions, additions, deletions and any combination thereof, preferably amino acid substitutions; and (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 86, 87, 89 and 90.

[0497] In another aspect, the anti-CD28 antigen binding domain comprises or consists essentially of:

[0498] (a) a heavy chain variable region (VH) comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO: 44, or a sequence having 80%, 85%, 90%, 95%, 97% or 99% sequence identity thereto; and (b) a light chain variable region (VH) comprising, or consisting of, an amino acid sequence as shown in SEQ ID NO: 45, or a sequence having 80%, 85%, 90%, 95%, 97% or 99% sequence identity thereto.

[0499] Fc domain

[0500] The Fc domain of the multifunctional molecule can form the heavy chain of the IgG immunoglobulin together with part of the antigen binding domain. In fact, the multifunctional molecule can specifically comprise a heavy chain, including a variable heavy chain (VH), CH1, hinge, CH2 and CH3 domains. However, the multifunctional molecule can also comprise other antigen binding domain structures, such as scFv covalently linked to the Fc domain.

[0501] The Fc domain may be a heavy chain constant domain from a human immunoglobulin heavy chain, for example, IgG1, IgG2, IgG3, IgG4 or other classes. Preferably, the multifunctional molecule comprises an IgG1 or IgG4 heavy chain constant domain.

[0502] The multifunctional molecule may in particular comprise a CH1 domain, a CH2 domain and a CH3 domain. For example, suitable heavy chain constant domains (CH1, CH2 and CH3) are provided as amino acid sequences SEQ ID NO: 23 and 24.

[0503] The multifunctional molecule preferably comprises an Fc domain. Such an Fc domain preferably comprises a first Fc chain and a second Fc chain, which are complementary and capable of dimerization. In certain embodiments, the amino acid sequence comprised by the first Fc chain differs from the amino acid sequence comprised by the second Fc chain in one or more amino acid additions, deletions or substitutions. Such Fc first and second Fc chains form a "heterodimer Fc". In other embodiments, the first and second Fc chains comprise the same amino acid sequence and form a "homodimer Fc".

[0504] Preferably, the Fc domain comprises CH2 and CH3 domains. Optionally, it may include all or part of the hinge region, CH2 domain and / or CH3 domain. In some aspects, the CH2 and / or CH3 domains are derived from human IgG4 or IgG1 heavy chains. Preferably, the Fc domain includes all or part of the hinge region. The hinge region may be derived from an immunoglobulin heavy chain, for example, IgG1, IgG2, IgG3, IgG4 or other classes. Preferably, the hinge region is derived from human IgG1, IgG2, IgG3, IgG4. More preferably, the hinge region is derived from human or humanized IgG1 or IgG4 heavy chains.

[0505] The IgG1 hinge region has three cysteines, two of which participate in the disulfide bond between the two heavy chains of the immunoglobulin. These identical cysteines allow efficient and consistent disulfide bonds to be formed between the Fc moieties. Therefore, the preferred hinge region of the present invention is derived from IgG1, more preferably derived from human IgG1. In some aspects, the first cysteine ​​in the hinge region of human IgG1 is mutated to another amino acid, preferably serine.

[0506] The hinge region of IgG4 may not be able to efficiently form interchain disulfide bonds. Suitable hinge regions for use in the present invention may be derived from IgG4 hinge regions, preferably containing mutations that enhance the correct formation of disulfide bonds between heavy chain derived portions (e.g., as described in Angal S, et al. (1993) Mol. Immunol., 30: 105-8). More preferably, the hinge region is derived from a human IgG4 heavy chain.

[0507] In an embodiment, the multifunctional molecule comprises a hinge region, in particular comprises or consists of the amino acid sequence shown in SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0508] The multifunctional molecule preferably comprises a dimeric Fc domain. Therefore, the multifunctional molecule comprises two Fc domains, and the Fc chains can form a dimeric Fc domain. The dimeric Fc domain can be a homodimer, and each Fc monomer (or chain) is identical or substantially identical. Alternatively, the dimeric Fc domain can be a heterodimer, and each Fc monomer (or chain) is different and complementary to promote the formation of a heterodimeric Fc domain.

[0509] More specifically, the Fc domain is a heterodimer Fc domain. The heterodimer Fc domain can be prepared by changing the amino acid sequence of each monomer (i.e., each Fc chain forming the Fc domain). The heterodimer Fc domain relies on different amino acid variants in the constant region on each chain to promote the formation of heterodimers and / or make heterodimers easier to purify than homodimers. There are multiple mechanisms for generating heterodimers of the present invention. In addition, as understood by professionals in the art, these mechanisms can be combined to ensure high heterodimerization. Therefore, the amino acid variants that lead to the generation of heterodimers are referred to as "heterodimerization variants". Heterodimerization variants can include steric hindrance variants (e.g., "knob-in-hole" or "tilted" variants described below and "charge pair" variants described below) and "π variants", which allow homodimers to be separated and purified from heterodimers. WO2014 / 145806, incorporated herein by reference in its entirety, discloses useful mechanisms of heterodimerization, including "knob-in-hole," "electrostatic steering" or "charge pair," π variants, and other general Fc variants. See also Ridgway et al., Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997 270:26; US Patent No. 8,216,805, Merchant et al., Nature Biotech. 16:677 (1998), all of which are incorporated herein by reference in their entirety. For "electrostatic steering," see Gunasekaran et al., J. Biol. Chem. 285(25): 19637 (2010), incorporated herein by reference in its entirety. For π variants, see US 2012 / 0149876, incorporated herein by reference in its entirety.

[0510] Then, in a preferred aspect, the heterodimeric Fc domain comprises a first Fc chain based on the "knob-hole" technology and a complementary second Fc chain. For example, the first Fc chain is a "knob" or K chain, which means that it comprises substitutions that characterize the knob chain; and the second Fc chain is a "hole" or H chain, which means that it comprises substitutions that characterize the hole chain. Vice versa, the first Fc chain is a "hole" or H chain, which means that it comprises substitutions that characterize the hole chain; and the second Fc chain is a "knob" or K chain, which means that it comprises substitutions that characterize the knob chain.

[0511] In a preferred aspect, the Fc chain associated with the anti-CD28 binding domain (ie, the second Fc chain) is a "hole" or H chain. Alternatively, the Fc chain associated with the anti-CD28 binding domain (ie, the second Fc chain) is a "knob" or K chain.

[0512] Optionally, the heterodimeric Fc domain may comprise one heterodimeric Fc chain comprising substitutions as shown in Table E below and another heterodimeric Fc chain comprising substitutions as shown in Table E below.

[0513] Table E (numbering according to EU index).

[0514]

[0515] In a preferred aspect, the first Fc chain is the "hole" or H chain and comprises the T366S / L368A / Y407V / Y349C substitutions and the second Fc chain is the "knob" or K chain and comprises the T366W / S354C substitutions.

[0516] Optionally, the Fc chain may further comprise additional substitutions.

[0517] In particular, for multifunctional molecules that target cell surface molecules, especially those on immune cells, it may be desirable to eliminate effector functions.Engineering Fc regions may also be desirable to reduce or enhance effector functions of multifunctional molecules.

[0518] In some aspects, amino acid modifications can be introduced into the Fc region to generate Fc region variants. In some aspects, Fc region variants have some (but not all) effector functions. Such multifunctional molecules are useful, for example, in applications where the half-life of antibodies in vivo is important, but certain effector functions are unnecessary or harmful. Many substitutions or replacements or deletions with altered effector functions are known in the art.

[0519] In one aspect, the constant region of the Fc domain contains mutations that reduce affinity for Fc receptors or reduce Fc effector function. For example, the constant region can contain mutations that eliminate glycosylation sites within the IgG heavy chain constant region. Preferably, the CH2 domain contains mutations that can eliminate glycosylation sites within the CH2 domain.

[0520] In a specific aspect, the Fc domain is modified to enhance binding to FcRn, thereby extending the half-life of the multifunctional molecule. In another or additional aspect, the Fc domain is modified to reduce binding to FcγR, thereby reducing ADCC or CDC, or enhance binding to FcγR, thereby increasing ADCC or CDC.

[0521] As shown in WO 01 / 58957, the change of amino acid near the junction of Fc part and non-Fc part significantly prolongs the serum half-life of Fc fusion protein. Therefore, relative to the naturally occurring sequence of immunoglobulin heavy chain and erythropoietin, the connecting region of protein or polypeptide of the present invention may contain changes, preferably within about 10 amino acids of the connecting point. These amino acid changes can cause hydrophobicity to increase. In one embodiment, the constant region is derived from IgG sequence, wherein the C-terminal lysine residue is replaced. Preferably, the C-terminal lysine of IgG sequence is replaced with non-lysine amino acid, such as alanine or leucine, to further prolong serum half-life.

[0522] In one embodiment, the constant region of the Fc domain has one or any combination of the mutations described in Table F below.

[0523] Table F: Suitable human engineered Fc domains of antibodies. The numbering of heavy chain constant region residues is according to EU numbering (Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969); www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html#refs)

[0524]

[0525]

[0526]

[0527] In a specific aspect, the multifunctional molecule comprises a human IgG1 heavy chain constant domain or an IgG1 Fc domain, optionally with a substitution or combination of substitutions selected from the group consisting of: T250Q / M428L; M252Y / S254T / T256E + H433K / N434F; E233P / L234V / L235A / G236A + A327G / A330S / P331S; E333A; S239D / A330L / I332E; P257I / Q311; K326W / E333S; S239D / I332E / G236A; N297A; L234A / L235A; P329G; N297A + M252Y / S254T / T256E; K322A, K444A, K444E, K444D, K444G, K444S, M428L, L309D, Q311H, N434S, M428L+N434S and L309D + Q311H + N434S, are preferably selected from the following group: N297A, optionally in combination with M252Y / S254T / T256E, and L234A / L235A, optionally in combination with P329G.

[0528] In another aspect, the multifunctional molecule comprises a human IgG4 heavy chain constant domain or a human IgG4 Fc domain, optionally with a substitution or combination of substitutions selected from the group consisting of S228P; L234A / L235A; L234A / L235A / P329G, P329G, S228P + M252Y / S254T / T256E, K444A K444E, K444D, K444G and K444S. Even more preferably, the multifunctional molecule, preferably a multifunctional molecule according to the invention, comprises an IgG4 Fc region with S228P that stabilizes IgG4.

[0529] As described herein, " / " and "+" refer to cumulative mutations. Thus, by mutations S228P + M252Y / S254T / T256E, it refers to the following mutations: S228P, M252Y, S254T, and T256E.

[0530] Typically, all subclasses of human IgG carry a C-terminal lysine residue (K444) of the antibody heavy chain, which is easily cut off during circulation. This cutting in the blood may damage or reduce the biological activity of the multifunctional molecule by releasing the immunostimulatory part connected to the multifunctional molecule. To avoid this problem, the K444 amino acid in the IgG domain can be replaced by another amino acid to reduce proteolytic cleavage, which is a mutation commonly used in antibodies. Then, in one aspect, the multifunctional molecule comprises at least one further amino acid replacement consisting of K444A, K444E, K444D, K444G or K444S, preferably K444A. In particular, the K444 amino acid in the IgG domain can be replaced by alanine to reduce proteolytic cleavage, which is a mutation commonly used in antibodies. Then, in one aspect, the multifunctional molecule comprises at least one further amino acid replacement consisting of K444A.

[0531] Optionally, the multifunctional molecule comprises an additional cysteine ​​residue in the C-terminal domain of the Fc domain to create an additional disulfide bond and possibly limit the flexibility of the multifunctional molecule.

[0532] In a specific aspect, the multifunctional molecule according to the invention comprises a heterodimer of an Fc domain comprising a "knob into hole" modification as described above. Preferably, such an Fc domain is an IgG1 or IgG4 Fc domain, e.g. as described above, even more preferably an IgG1 Fc domain comprising an N297A mutation, e.g. as described above.

[0533] In some embodiments, the Fc chain linked to the first binding moiety, in particular the Fc chain linked to the anti-PD1 binding domain, may comprise or consist of an amino acid sequence as shown in SEQ ID NO: 25 or 26, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0534] In some embodiments, the Fc chain linked to the anti-CD28 binding domain is a "hole" chain or H chain and comprises T366S / L368A / Y407V / Y349C substitutions and optionally N297A, and the other Fc chain is a "knob" chain or K chain and comprises T366W / S354C substitutions and optionally N297A. Preferably, the Fc chain linked to the anti-CD28 binding domain is a "hole" chain or H chain and comprises or consists of the amino acid sequence as shown in SEQ ID NO: 54.

[0535] More specifically, the multifunctional molecule may comprise an Fc domain comprising or consisting of:

[0536] i) a first Fc chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 25 (knob), optionally having one, two or three modifications selected from substitution, addition, deletion and any combination thereof.

[0537] ii) a second Fc chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 26 or 54 (hole), optionally having one, two or three modifications selected from substitution, addition, deletion and any combination thereof.

[0538] In a first embodiment, the multifunctional molecule comprises an anti-CD28 binding domain linked to the C-terminus of an Fc domain, which is a holmium chain.

[0539] In a second embodiment, the multifunctional molecule comprises an anti-CD28 binding domain linked to the N-terminus of an Fc domain, said Fc domain being a holmium chain.

[0540] In a third embodiment, the multifunctional molecule comprises an anti-CD28 binding domain linked to the C-terminus of an Fc domain, which is the knob chain.

[0541] In a fourth embodiment, the multifunctional molecule comprises an anti-CD28 binding domain linked to the N-terminus of an Fc domain, which is the knob chain.

[0542] Peptide linker

[0543] The present invention includes multifunctional molecules which may comprise one or more peptide linkers.

[0544] The peptide linker is usually of sufficient length and flexibility to ensure that the two protein elements connected in the middle by the peptide linker have sufficient spatial freedom to perform their functions and avoid the formation of α-helix and β-fold that affects the stability of the recombinant multifunctional molecule.

[0545] As used herein, the term "peptide linker" refers to a sequence of at least one amino acid that connects different domains or chains of a multifunctional molecule. Such linkers help prevent steric hindrance. The length of the linker is generally 3-44 amino acid residues. Preferably, the linker has 3-30 amino acid residues. In some embodiments, the linker has 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues.

[0546] The linker sequence can be a naturally occurring sequence or a non-naturally occurring sequence. If used for therapeutic purposes, the linker is preferably non-immunogenic in the subject to which the multifunctional molecule is administered. One group of useful linker sequences is a linker derived from the hinge region of the heavy chain antibody described in WO96 / 34103 and WO 94 / 04678. Other examples are polyalanine linker sequences. Further preferred examples of linker sequences are Gly / Ser linkers of different lengths, including (Gly4Ser)4, (Gly4Ser)3, (Gly4Ser)2, Gly4Ser, Gly3Ser, Gly3, Gly2ser and (Gly3Ser2)3, particularly (Gly4Ser)3.

[0547] In one embodiment, the one or more linkers comprised in the multifunctional molecule are selected from the group consisting of (Gly4Ser)4, (Gly4Ser)3, (Gly4Ser)2, Gly4Ser, Gly3Ser, Gly3, Gly2ser and (Gly3Ser2)3, preferably (Gly4Ser)3.

[0548] In one embodiment, the one or more linkers contained in the multifunctional molecule are selected from the following group: (GGGGS)4, (GGGGS)3, (GGGGS)2, GGGGS, (GGGS)3, (GGGS)2, GGGS, GGG, GGS and GG, and preferably (GGGGS)3.

[0549] In an embodiment, the anti-CD28 binding moiety is a scFv and it is covalently linked to an Fc domain, in particular to the N- or C-terminus of an Fc domain such as described herein, via a peptide linker such as described above, in particular a (GGGGS)3 linker.

[0550] In an embodiment, the C-terminus of the VL domain of the anti-CD28 antigen binding domain can be linked to the N-terminus of the Fc domain via a peptide linker, such as described above, in particular a (GGGGS) 3 linker.

[0551] Alternatively, the C-terminus of the VH domain of the anti-CD28 antigen-binding domain is linked to the N-terminus of the Fc domain via a peptide linker such as described above, in particular a (GGGGS) 3 linker.

[0552] In an embodiment, the N-terminus of the VL domain of the anti-CD28 antigen binding domain can be linked to the C-terminus of the Fc domain via a peptide linker, such as described above, in particular a (GGGGS) 3 linker.

[0553] Alternatively, the N-terminus of the VH domain of the anti-CD28 antigen-binding domain is linked to the C-terminus of the Fc domain via a peptide linker such as described above, in particular a (GGGGS) 3 linker.

[0554] In an embodiment, the anti-CD28 binding moiety is a scFv and is covalently linked to the CL domain of the first binding moiety via a peptide linker, e.g., as described above, particularly a (GGGGS)3 linker.

[0555] In an embodiment, the C-terminus of the VL of the anti-CD28 antigen binding domain can be connected to the N-terminus of the CL domain of the first binding moiety via a peptide linker, such as described above, particularly a (GGGGS)3 linker, as described in SEQ ID NO: 57.

[0556] Alternatively, the C-terminus of the VH of the anti-CD28 antigen binding domain is linked to the N-terminus of the CL domain of the first binding moiety via a peptide linker, such as described above, in particular a (GGGGS)3 linker, as depicted in SEQ ID NO: 57.

[0557] In an embodiment, the N-terminus of the VL of the anti-CD28 antigen binding domain can be connected to the C-terminus of the CL domain of the first binding moiety via a peptide linker, such as described above, particularly a (GGGGS)3 linker, as described in SEQ ID NO: 57.

[0558] Alternatively, the N-terminus of the VH of the anti-CD28 antigen binding domain is connected to the C-terminus of the CL domain of the first binding moiety via a peptide linker, such as described above, in particular a (GGGGS)3 linker, as depicted in SEQ ID NO: 57.

[0559] In an embodiment, wherein the anti-CD28 antigen binding domain is a scFv, and the VL and VH domains of the scFv are preferably covalently linked to each other via a peptide linker, such as described above, in particular a (GGGGS)3 linker, as depicted in SEQ ID NO: 57.

[0560] In particular, the C-terminus of the VL of the anti-CD28 antigen binding domain is connected to the N-terminus of the VH of the anti-CD28 antigen binding domain via a peptide linker, such as described above, in particular a (GGGGS)3 linker, as depicted in SEQ ID NO: 57.

[0561] Alternatively, the C-terminus of the VH of the anti-CD28 antigen binding domain is connected to the N-terminus of the VL of the anti-CD28 antigen binding domain via a peptide linker, such as described above, in particular a (GGGGS)3 linker, as depicted in SEQ ID NO: 57.

[0562] Thus, in some aspects, the anti-CD28 binding domain is a scFV comprising, from N-terminus to C-terminus: a VH domain of SEQ ID NO: 44, a peptide linker of SEQ ID NO: 57, and a VL domain of SEQ ID NO: 45. Alternatively, the anti-CD28 binding domain is a scFV comprising, from N-terminus to C-terminus: a VL domain of SEQ ID NO: 45, a peptide linker of SEQ ID NO: 57, and a VH domain of SEQ ID NO: 44.

[0563] At the joining junction, the C-terminal lysine residue of VL or VH can be mutated to alanine to reduce proteolytic cleavage.

[0564] Thus, the multifunctional molecule comprises a first binding moiety that binds to a target specifically expressed on the surface of a T cell, linked via its C-terminus to the N-terminus of a first Fc chain, an anti-CD28 binding domain, optionally covalently linked via its C-terminus to the N-terminus of a second Fc chain via a peptide linker, wherein the first Fc chain and the second Fc chain are complementary. Preferably, the anti-CD28 binding domain is a scFv, such that the molecule may further comprise a peptide linker between the VL and VH domains of the scFv.

[0565] In another embodiment, the multifunctional molecule comprises a first binding moiety that binds to a target specifically expressed on the surface of a T cell, linked via its C-terminus to the N-terminus of a first Fc chain, an anti-CD28 binding domain, optionally covalently linked via its N-terminus to the C-terminus of said first Fc chain via a peptide linker, and a complementary second Fc chain that does not comprise a binding moiety. Preferably, the anti-CD28 binding domain is a scFv, such that the molecule may further comprise a peptide linker between the VL and VH domains of said scFv.

[0566] In another embodiment, the multifunctional molecule comprises an Fc domain connected to two first binding moieties, the first binding moiety binding to a target specifically expressed on the surface of T cells and comprising VH and VL domains, and an anti-CD28 binding domain, optionally via a peptide linker, covalently linked via its C-terminus to the N-terminus of the light chain of the first binding moiety, or covalently linked via its N-terminus to the C-terminus of the Fc domain or to the C-terminus of the light chain of the first binding moiety. Preferably, the anti-CD28 binding domain is an scFv, such that the molecule further comprises a peptide linker between the VL and VH domains of said scFv.

[0567] Examples of multifunctional molecules

[0568] Specific examples of multifunctional molecules according to the present invention are provided herein,

[0569] The multifunctional molecule comprises or consists of the following:

[0570] - a first binding moiety, which is an anti-PD1 binding moiety, in particular an anti-PD1 binding domain selected from the group consisting of Fab, Fab', F(ab')2, CrossMAb or single chain (scFv), said anti-PD1 binding moiety preferably having the amino acid sequence described above under the section "First binding moiety";

[0571] - a second binding moiety, which is an anti-CD28 binding moiety with agonist activity, in particular an antigen binding domain selected from the group consisting of Fab, Fab', CrossMAb or single chain (scFv), said anti-CD28 binding moiety preferably having the amino acid sequence described above under the section "Second binding moiety"; and

[0572] - a first and a second Fc chain, said first and second Fc chains being complementary and together forming an Fc domain;

[0573] And wherein the multifunctional molecule comprises a single anti-CD28 binding site.

[0574] Figure 1 Specific examples of multifunctional molecules are provided in, configurations A1, B1, B2, B3, B4, C1, C2, C3, C4, C5 and C6.

[0575] Preferably, the bifunctional molecule has the B1 configuration, in particular Fig. 7A In particular, the B1 configuration consists essentially of: i) an anti-PD-1 Fab linked to a first Fc chain and ii) an anti-CD28 scFv linked to a second Fc chain; the first and second Fc chains are complementary so as to form an Fc domain.

[0576] In other words, the multifunctional molecule binds CD28 monovalently and is unable to cross-link CD28.

[0577] In all and any specific examples of multifunctional molecules disclosed in this section and comprising an anti-PD1 binding portion as a first binding portion, the anti-PD1 binding portion may be replaced by another binding portion that binds to another target specifically expressed on the surface of T cells (particularly, for example, CTLA-4, BTLA, VISTA, TIGIT, CD160, LAG3 and TIM3).

[0578] In a first aspect, the multifunctional molecule comprises or consists of:

[0579] - a first binding moiety which is an anti-PD1 Fab linked to a first Fc chain,

[0580] - a second binding moiety; which is an anti-CD28 agonist Fab or CrossMab linked to a second Fc chain; and

[0581] - said first and second Fc chains are complementary and together form an Fc domain;

[0582] And the multifunctional molecule comprises a single anti-CD28 binding portion.

[0583] More specifically, the multifunctional molecule comprises or consists of:

[0584] - a first chain, which comprises or consists of the VL and CL domains of the anti-PD1 binding moiety from N-terminus to C-terminus;

[0585] - a second chain, which comprises or consists of, from N-terminus to C-terminus: the VH and CH1 domains of an anti-PD1 binding moiety, optionally a hinge, the CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming the first Fc chain,

[0586] - a third chain, which comprises or consists of, from N-terminus to C-terminus: (i) (Fab) the VH and CH1 domains of an anti-CD28 binding moiety, optionally a hinge, the CH2 and CH3 domains of an antibody, the CH2 and CH3 domains together forming a second Fc chain; or (ii) (CrossMAb) the VH and CL domains of an anti-CD28 binding moiety, optionally a hinge, the CH2 and CH3 domains of an antibody, the CH2 and CH3 domains together forming a second Fc chain,

[0587] - a fourth chain which, from N-terminus to C-terminus, comprises or consists of: (i) the VL and CL domains of the (Fab) anti-CD28 binding moiety; or (ii) the VL and CH1 domains of the (CrossMAb) anti-CD28 binding moiety,

[0588] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0589] In particular, the first Fc chain is the "knob", and the second Fc chain is the "hole". Alternatively, the first Fc chain is the "hole", and the second Fc chain is the "knob".

[0590] Preferably, the multifunctional molecule comprises or consists of:

[0591] - a first chain, which is a light chain of an anti-PD1 antibody and comprises or consists of, from N-terminus to C-terminus: a VL and CL domain comprising or consisting of SEQ ID NOs: 16 and 18, respectively, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof;

[0592] - a second chain, which is a heavy chain of an anti-PD1 antibody and comprises or consists of, from N-terminus to C-terminus: (i) a VL and CH1 domain comprising or consisting of SEQ ID NOs: 15 and 17, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (ii) a hinge, optionally preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (iii) CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming a first Fc chain, comprising or consisting of SEQ ID NO: 25, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof;

[0593] - a third chain, which is a heavy chain of an anti-CD28 antibody and comprises or consists of, from N-terminus to C-terminus: (i) a VL and CH1 domain comprising or consisting of SEQ ID NOs: 44 and 46, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (ii) a hinge, optionally preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (iii) CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming a second Fc chain, which comprises or consists of SEQ ID NO: 54, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof;

[0594] - a fourth chain, which is a light chain of an anti-CD28 antibody and comprises or consists of, from N-terminus to C-terminus: the VL and CL domains of an anti-CD28 antibody comprising or consisting of SEQ ID NOs: 45 and 47, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof,

[0595] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0596] Alternatively, the multifunctional molecule comprises or consists of:

[0597] - a first chain, which is a light chain of an anti-PD1 antibody and comprises or consists of, from N-terminus to C-terminus: a VL and CL domain comprising or consisting of ID NO: NO: 16 and 18, respectively, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof;

[0598] - a second chain, which is a heavy chain of an anti-PD1 antibody and comprises or consists of the following from N-terminus to C-terminus: (i) VL and CH1 domains comprising or consisting of SEQ ID NOs: 15 and 17, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (ii) a hinge, optionally preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (iii) CH2 and CH3 domains, said CH2 and CH3 domains forming a first Fc chain, which comprises or consists of SEQ ID NO: 25, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof;

[0599] - a third chain, which is a heavy chain of an anti-CD28 antibody and comprises or consists of, from N-terminus to C-terminus: (i) VH and CL domains comprising or consisting of SEQ ID NOs: 44 and 47, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; ii) a hinge, optionally preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (iii) CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming a second Fc chain, which comprises or consists of SEQ ID NO: 54, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof;

[0600] - a fourth chain, which is a light chain of an anti-CD28 antibody and comprises or consists of, from N-terminus to C-terminus: a VL and CH1 domain of an anti-CD28 antibody comprising or consisting of SEQ ID NOs: 45 and 46, respectively, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof,

[0601] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0602] In a very specific embodiment, the multifunctional molecule comprises or consists of:

[0603] - a first strand comprising or consisting of the sequence as shown in SEQ ID NO: 20, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof;

[0604] - a second strand comprising or consisting of the sequence as shown in SEQ ID NO: 27, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof;

[0605] - a third strand comprising or consisting of the sequence as shown in SEQ ID NO: 53, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, and

[0606] - a fourth strand comprising or consisting of the sequence as shown in SEQ ID NO: 51, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0607] In a second aspect, the multifunctional molecule comprises or consists of:

[0608] - a first binding moiety which is an anti-PD1 Fab, Fab' or CrossMAb;

[0609] - a second binding moiety which is an agonistic anti-CD28 scFV; and

[0610] - a first Fc chain and a second Fc chain, said first and second Fc chains being complementary and together forming an Fc domain.

[0611] In one embodiment, the multifunctional molecule comprises or consists in particular of:

[0612] - a first chain, which comprises or consists of the VL and CL domains of an anti-PD1 antibody from the N-terminus to the C-terminus,

[0613] a second chain comprising or consisting of, from N-terminus to C-terminus: a VH and a CH1 domain, optionally a hinge, a CH2 and a CH3 domain of an antibody, said CH2 and CH3 domains together forming a first Fc chain,

[0614] a third chain comprising or consisting of, from N-terminus to C-terminus: the VL and VH domains of an anti-CD28 antibody connected by a peptide linker, optionally a peptide linker, optionally a hinge, the CH2 and CH3 domains of an antibody, said CH2 and CH3 domains together forming a second Fc chain,

[0615] Therein the first and second Fc chains are complementary and thereby together form an Fc domain.

[0616] In particular, the first Fc chain is the "knob", and the second Fc chain is the "hole". Alternatively, the first Fc chain is the "hole", and the second Fc chain is the "knob".

[0617] Preferably, the multifunctional molecule comprises or consists of:

[0618] - a first chain, which is a light chain of an anti-PD1 antibody and comprises or consists of, from N-terminus to C-terminus: a VL and CL domain comprising or consisting of SEQ ID NOs: 16 and 18, respectively, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof;

[0619] - a second chain, which is a heavy chain of an anti-PD1 antibody and comprises or consists of, from N-terminus to C-terminus: (i) VL and CH1 domains comprising or consisting of SEQ ID NOs: 15 and 17, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (ii) a hinge, optionally preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (iii) CH2 and CH3 domains, said CH2 and CH3 domains forming a first Fc chain, comprising or consisting of SEQ ID NO: 25, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; and

[0620] a third chain comprising or consisting of: (i) an anti-CD28 scFv comprising the VL and VH domains of an anti-CD28 antibody, the VL and VH domains comprising or consisting of SEQ ID NO: 45 and 44, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, the VL and VH domains preferably being connected via a peptide linker, preferably SEQ ID NO: 57 (ii) optionally a peptide linker, (iii) optionally a hinge, preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof iv) the CH2 and CH3 domains of an antibody, the CH2 and CH3 domains forming a second Fc chain comprising or consisting of SEQ ID NO: 26 or 54, preferably SEQ ID NO: 54, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof;

[0621] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0622] In a very specific embodiment, the multifunctional molecule comprises or consists of:

[0623] - a first strand comprising or consisting of a sequence as shown in SEQ ID NO: 20, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof

[0624] - a second strand comprising or consisting of the sequence as shown in SEQ ID NO: 27, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, and

[0625] - a third strand comprising or consisting of the sequence as shown in SEQ ID NO: 56, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0626] In a preferred embodiment, the multifunctional molecule of the present invention comprises or consists of:

[0627] - a first chain comprising or consisting of a sequence as shown in SEQ ID NO: 20, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; preferably amino acid substitutions; said modifications are preferably in the framework regions;

[0628] - a second chain comprising or consisting of a sequence as shown in SEQ ID NO: 27, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, preferably amino acid substitutions; said modifications are preferably in the framework regions; and

[0629] - a third chain, which comprises or consists of a sequence as shown in SEQ ID NO: 56, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; preferably amino acid substitutions; said modifications are preferably in the framework regions.

[0630] In one embodiment, the multifunctional molecule comprises or consists in particular of:

[0631] - a first chain, which comprises or consists of the VL and CL domains of an anti-PD1 antibody from N-terminus to C-terminus;

[0632] - a second chain, which comprises or consists of, from N-terminus to C-terminus: the VH and CH1 domains of an anti-PD1 antibody, optionally a hinge, the CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming a first Fc chain; and

[0633] - a third chain, which comprises or consists of, from N-terminus to C-terminus: the VH and VL domains of an anti-CD28 antibody, preferably the VL and VH domains are connected by a peptide linker, optionally a peptide linker, optionally a hinge, the CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming a second Fc chain;

[0634] Therein the first and second Fc chains are complementary and thereby together form an Fc domain.

[0635] In particular, the first Fc chain is the "knob", and the second Fc chain is the "hole". Alternatively, the first Fc chain is the "hole", and the second Fc chain is the "knob".

[0636] Preferably, the multifunctional molecule comprises or consists of:

[0637] - a first chain, which is a light chain of an anti-PD1 antibody and comprises or consists of, from N-terminus to C-terminus: a VL and CL domain comprising or consisting of SEQ ID NOs: 16 and 18, respectively, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof;

[0638] - a second chain, which is a heavy chain of an anti-PD1 antibody and comprises or consists of, from N-terminus to C-terminus: (i) VH and CH1 domains comprising or consisting of SEQ ID NOs: 15 and 17, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (ii) optionally a hinge, preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof (iii) CH2 and CH3 domains of an antibody, the CH2 and CH3 domains forming a first Fc chain, which comprises or consists of SEQ ID NO: 25, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; and

[0639] a third chain comprising or consisting of, from N-terminus to C-terminus: (i) an anti-CD28 scFv comprising the VH and VL domains of an anti-CD28 antibody, the VL and VH domains comprising or consisting of SEQ ID NO: 44 and 45, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, the VL and VH domains preferably being connected via a peptide linker, preferably SEQ ID NO: 57 (ii) optionally a peptide linker, preferably SEQ ID NO: 57, (iii) optionally a hinge, preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof iv) the CH2 and CH3 domains of an antibody, the CH2 and CH3 domains forming a second Fc chain, comprising or consisting of SEQ ID NO: 26 or 54, preferably SEQ ID NO: 54, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof;

[0640] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0641] In a very specific embodiment, the multifunctional molecule comprises or consists of:

[0642] - a first strand comprising or consisting of a sequence as shown in SEQ ID NO: 20, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof

[0643] - a second strand comprising or consisting of the sequence as shown in SEQ ID NO: 27, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, and

[0644] - a third strand comprising or consisting of the sequence as shown in SEQ ID NO: 55, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0645] Preferably, one, two or three modifications are preferably outside the CDRs (ie, within the framework regions).

[0646] In one embodiment, the multifunctional molecule comprises or consists in particular of:

[0647] a first chain comprising or consisting of, from N-terminus to C-terminus: i) optionally a hinge, ii) the CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming a first Fc chain,

[0648] - a second chain comprising or consisting of, from N-terminus to C-terminus: (i) the VH and CH1 domains of an anti-PD1 antibody, optionally a hinge, the CH2 and CH3 domains of the antibody, said CH2 and CH3 domains forming a second Fc chain, (ii) optionally a peptide linker, and (iii) an anti-CD28 scFV comprising the VL and VH domains of an anti-CD28 antibody, preferably connected by a peptide linker; and

[0649] - a third chain, which comprises or consists of the VL and CL domains of an anti-PD1 antibody from the N-terminus to the C-terminus,

[0650] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0651] In particular, the first Fc chain is the "knob", and the second Fc chain is the "hole". Alternatively, the first Fc chain is the "hole", and the second Fc chain is the "knob".

[0652] Preferably, the multifunctional molecule comprises or consists of:

[0653] - a first chain, which is the Fc chain of an antibody and comprises or consists of, from N-terminus to C-terminus: i) optionally a hinge, preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof ii) CH2 and CH3 domains of an antibody, the CH2 and CH3 domains forming the first Fc chain, which comprises or consists of SEQ ID NO: 25, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof;

[0654] - A second chain, which comprises or consists of the following from N-terminus to C-terminus: a) a heavy chain of an anti-PD1 antibody, comprising (i) VH and CH1 domains comprising or consisting of SEQ ID NOs: 15 and 17, respectively, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof; (ii) optionally a hinge, preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof (iii) CH2 and CH3 domains, which form a second Fc chain, which comprises or consists of SEQ ID NO: 26, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof; (b) optionally a peptide linker, preferably SEQ ID NO: 57 (c) an anti-CD28 scFv comprising the VL and VH domains of an anti-CD28 antibody, the VL and VH domains comprising SEQ ID NO: 45 and 44 or consisting thereof, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof, preferably connected via a peptide linker, preferably SEQ ID NO: 57, and

[0655] - a third chain, which is a light chain of an anti-PD1 antibody and comprises or consists of, from N-terminus to C-terminus: a VL and CL domain comprising or consisting of SEQ ID NOs: 16 and 18, respectively, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof;

[0656] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0657] In a very particular embodiment, the multifunctional molecule comprises or consists of:

[0658] - a first strand comprising or consisting of a sequence as shown in SEQ ID NO: 25, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof

[0659] - a second strand comprising or consisting of the sequence as shown in SEQ ID NO: 62, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, and

[0660] - a third strand comprising or consisting of the sequence as shown in SEQ ID NO: 20, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0661] In this aspect, the first Fc chain, in particular the knob chain, preferably does not contain any other binding moieties or immunotherapeutic agents.

[0662] In one embodiment, the multifunctional molecule comprises or consists in particular of:

[0663] a first chain comprising or consisting of, from N-terminus to C-terminus: i) optionally a hinge, ii) the CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming a first Fc chain,

[0664] - a second chain comprising or consisting of, from N-terminus to C-terminus: (i) the VH and CH1 domains of an anti-PD1 antibody, a hinge, the CH2 and CH3 domains of an anti-PD1 antibody, said CH2 and CH3 domains forming a second Fc chain, optionally a peptide linker, and (ii) an anti-CD28 scFv comprising the VH and VL domains of an anti-CD28 antibody, preferably connected by a peptide linker; and

[0665] - a third chain, which comprises or consists of the VL and CL domains of an anti-PD1 antibody from the N-terminus to the C-terminus,

[0666] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0667] In particular, the first Fc chain is the "knob", and the second Fc chain is the "hole". Alternatively, the first Fc chain is the "hole", and the second Fc chain is the "knob".

[0668] In this aspect, the first Fc chain, in particular the knob chain, preferably does not contain any other binding moieties or immunotherapeutic agents.

[0669] Preferably, the multifunctional molecule comprises or consists of:

[0670] - a first chain, which is the first Fc chain of an antibody and comprises or consists of: i) optionally a hinge, preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof ii) CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming the first Fc chain, which comprises or consists of SEQ ID NO: 25, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; said first Fc chain does not contain any other binding moiety or immunotherapeutic agent;

[0671] - a second chain comprising or consisting of: a) a heavy chain of an anti-PD1 antibody comprising (i) VH and CH1 domains comprising or consisting of SEQ ID NOs: 15 and 17, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (ii) optionally a hinge, preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof (iii) CH2 and CH3 domains of an antibody, the CH2 and CH3 domains forming a second Fc chain comprising or consisting of SEQ ID NO: 26, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (b) optionally a peptide linker, preferably SEQ ID NO: 57, and (c) an anti-CD28 scFv comprising the VH and VL domains of an anti-CD28 antibody, the VH and VL domains comprising SEQ ID NO: 44 and 45 or consisting thereof, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof, preferably connected via a peptide linker, preferably SEQ ID NO: 57, and

[0672] - a third chain, which is a light chain of an anti-PD1 antibody and comprises or consists of: a VL and CL domain comprising or consisting of SEQ ID NOs: 16 and 18, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof;

[0673] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0674] In a very specific embodiment, the multifunctional molecule comprises or consists of:

[0675] - a first strand comprising or consisting of a sequence as shown in SEQ ID NO: 25, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof

[0676] - a second strand comprising or consisting of the sequence as shown in SEQ ID NO: 61, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, and

[0677] - a third strand comprising or consisting of the sequence as shown in SEQ ID NO: 20, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0678] In a third aspect, the multifunctional molecule comprises or consists of:

[0679] - a first binding moiety which is an anti-PD1 F(ab')2;

[0680] - a second binding moiety which is an agonistic anti-CD28 scFV;

[0681] - a first and a second Fc chain, said first and second Fc chains being complementary and forming an Fc domain,

[0682] The anti-PD1 F(ab')2 is linked to the Fc domain.

[0683] Preferably, the F(ab')2 comprises a Fab and a CrossMAb, one chain of each of which is linked to Fc.

[0684] In other words, the multifunctional molecule may comprise or consist of:

[0685] - a first binding moiety which is an anti-PD1 antibody; and

[0686] - A second binding moiety which is an anti-CD28 scFV linked to an anti-PD1 antibody, optionally via a peptide linker.

[0687] Preferably, the anti-PD1 antibody comprises a CrossMAb, preferably one CrossMAb.

[0688] Optionally, the anti-CD28 scFV is linked to

[0689] a) the C-terminus of a light chain, preferably one light chain;

[0690] b) the N-terminus of a light chain, preferably one light chain; or

[0691] c) the C-terminus of a heavy chain, preferably one heavy chain.

[0692] In one embodiment, the multifunctional molecule comprises or consists in particular of:

[0693] - a first chain which comprises or consists of, from N-terminus to C-terminus: (i) the VL and CH1 domains of an anti-PD1 antibody, (ii) optionally a peptide linker, and (iii) an anti-CD28 scFv comprising the VH and VL domains of an anti-CD28 antibody, preferably connected via a peptide linker,

[0694] - a second chain, which comprises or consists of, from N-terminus to C-terminus: the VH and CL domains of an anti-PD1 antibody, optionally a hinge, the CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming the first Fc chain,

[0695] - a third chain, which comprises or consists of, from N-terminus to C-terminus: the VH and CH1 domains of an anti-PD1 antibody, optionally a hinge, the CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming a second Fc chain,

[0696] - a fourth chain, which comprises or consists of (i) the VL and CL domains of an anti-PD1 antibody from N-terminus to C-terminus;

[0697] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0698] In particular, the first Fc chain is the "knob" and the second Fc chain is the "hole". Alternatively, the first Fc chain is the "hole" or the second Fc chain is the "knob".

[0699] Preferably, the multifunctional molecule comprises or consists of:

[0700] - A first chain comprising or consisting of: (i) a chain comprising, from N-terminus to C-terminus, the VL and CH1 domains of an anti-PD1 antibody, the VL and CH1 domains comprising or consisting of SEQ ID NOs: 16 and 17, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof (ii) optionally a peptide linker, preferably SEQ ID NO: 57 (iii) an anti-CD28 scFV comprising the VH and VL domains of an anti-CD28 antibody, the VH and VL domains comprising or consisting of SEQ ID NOs: 44 and 45, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, preferably connected via a peptide linker, preferably SEQ ID NO: 57;

[0701] - a second chain, which comprises or consists of the following from N-terminus to C-terminus: (i) VH and CL domains of an anti-PD1 antibody comprising or consisting of SEQ ID NOs: 15 and 18, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (ii) optionally a hinge, preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof (iii) CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming a first Fc chain, which comprises or consists of SEQ ID NO: 25, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof;

[0702] - a third chain comprising or consisting of a heavy chain of an anti-PD1 antibody and comprising (i) a VH and CH1 domain comprising or consisting of SEQ ID NOs: 15 and 17, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (ii) optionally a hinge, preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof (iii) CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming a second Fc chain, comprising or consisting of SEQ ID NO: 26, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof;

[0703] - a fourth chain comprising or consisting of: (i) a light chain of an anti-PD1 antibody comprising: a VL and CL domain comprising or consisting of SEQ ID NOs: 16 and 18, respectively, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof;

[0704] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0705] In a very specific embodiment, the multifunctional molecule comprises or consists of:

[0706] - a first strand comprising or consisting of the sequence as shown in SEQ ID NO: 60, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof

[0707] - a second strand comprising or consisting of the sequence as shown in SEQ ID NO: 28, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof

[0708] - a third strand comprising or consisting of the sequence as shown in SEQ ID NO: 29, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, and

[0709] - a fourth strand comprising or consisting of the sequence as shown in SEQ ID NO: 20, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0710] In one embodiment, the multifunctional molecule comprises or consists in particular of:

[0711] a first chain comprising or consisting of, from N-terminus to C-terminus: (i) an anti-CD28 scFv comprising the VH and VL domains of an anti-CD28 antibody, preferably connected via a peptide linker (ii) optionally a peptide linker, and (iii) the VL and CH1 domains of an anti-PD1 antibody,

[0712] - a second chain, which comprises or consists of, from N-terminus to C-terminus: the VH and CL domains of an anti-PD1 antibody, optionally a hinge, the CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming the first Fc chain,

[0713] - a third chain, which comprises or consists of, from N-terminus to C-terminus: the VH and CH1 domains of an anti-PD1 antibody, optionally a hinge, the CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming a second Fc chain,

[0714] - a fourth chain, which comprises or consists of (i) the VL and CL domains of an anti-PD1 antibody from N-terminus to C-terminus;

[0715] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0716] In particular, the first Fc chain is the "knob", and the second Fc chain is the "hole". Alternatively, the first Fc chain is the "hole", and the second Fc chain is the "knob".

[0717] Preferably, the multifunctional molecule comprises or consists of:

[0718] - a first chain comprising or consisting of: (i) an anti-CD28 scFv comprising a VH and VL domain comprising or consisting of SEQ ID NOs: 44 and 45, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, preferably connected via a peptide linker, preferably SEQ ID NO: 57 (ii) optionally a peptide linker, preferably SEQ ID NO: 57 (iii) a chain comprising, from N-terminus to C-terminus, the VL and CH1 domains of an anti-PD1 antibody comprising or consisting of SEQ ID NOs: 16 and 17, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof;

[0719] - a second chain, which comprises or consists of the following from N-terminus to C-terminus: (i) VH and CL domains of an anti-PD1 antibody comprising or consisting of SEQ ID NOs: 15 and 18, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (ii) optionally a hinge, preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof (iii) CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming a first Fc chain, which comprises or consists of SEQ ID NO: 25, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof;

[0720] - a third chain comprising or consisting of a heavy chain of an anti-PD1 antibody and comprising, from N-terminus to C-terminus, (i) a VH and CH1 domain comprising or consisting of SEQ ID NOs: 15 and 17, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (ii) optionally a hinge, preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof (iii) CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming a second Fc chain, comprising or consisting of SEQ ID NO: 26, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; and

[0721] - a fourth chain comprising or consisting of: (i) a light chain of an anti-PD1 antibody, comprising, from N-terminus to C-terminus, VL and CL domains comprising or consisting of SEQ ID NOs: 16 and 18, respectively, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof;

[0722] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0723] In a very specific embodiment, the multifunctional molecule comprises or consists of:

[0724] - a first strand comprising or consisting of a sequence as shown in SEQ ID NO: 59, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof

[0725] - a second strand comprising or consisting of a sequence as shown in SEQ ID NO: 28, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof

[0726] - a third strand comprising or consisting of the sequence as shown in SEQ ID NO: 29, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, and

[0727] - a fourth strand comprising or consisting of the sequence as shown in SEQ ID NO: 20, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0728] In one embodiment, the multifunctional molecule comprises or consists in particular of:

[0729] - a first chain which comprises or consists of, from N-terminus to C-terminus: (i) the VL and CH1 domains of an anti-PD1 antibody, (ii) optionally a peptide linker, and (iii) an anti-CD28 scFv comprising the VL and VH domains of an anti-CD28 antibody, preferably connected via a peptide linker,

[0730] - a second chain, which comprises or consists of, from N-terminus to C-terminus: the VH and CL domains of an anti-PD1 antibody, optionally a hinge, the CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming the first Fc chain,

[0731] - a third chain, which comprises or consists of, from N-terminus to C-terminus: the VH and CH1 domains of an anti-PD1 antibody, optionally a hinge, the CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming a second Fc chain, and

[0732] - a fourth chain, which comprises or consists of (i) the VL and CL domains of an anti-PD1 antibody from N-terminus to C-terminus;

[0733] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0734] In particular, the first Fc chain is the "knob", and the second Fc chain is the "hole". Alternatively, the first Fc chain is the "hole", and the second Fc chain is the "knob".

[0735] Preferably, the multifunctional molecule comprises or consists of:

[0736] - a first chain comprising or consisting of: (i) a chain comprising, from N-terminus to C-terminus, the VL and CH1 domains of an anti-PD1 antibody, the VL and CH1 domains comprising or consisting of SEQ ID NOs: 16 and 17, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof (ii) optionally a peptide linker, preferably SEQ ID NO: 57 and (iii) an anti-CD28 scFV comprising the VL and VH domains comprising or consisting of SEQ ID NOs: 45 and 44, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, preferably connected via a peptide linker, preferably SEQ ID NO: 57;

[0737] - a second chain, which comprises or consists of the following from N-terminus to C-terminus: (i) VH and CL domains of an anti-PD1 antibody comprising or consisting of SEQ ID NOs: 15 and 18, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (ii) optionally a hinge, preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof (iii) CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming a first Fc chain, which comprises or consists of SEQ ID NO: 25, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof;

[0738] - a third chain comprising or consisting of a heavy chain and comprising, from N-terminus to C-terminus, (i) VH and CH1 domains of an anti-PD1 antibody comprising or consisting of SEQ ID NOs: 15 and 17, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (ii) optionally a hinge, preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, and (iii) CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming a second Fc chain comprising or consisting of SEQ ID NO: 26, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; and

[0739] - a fourth chain comprising or consisting of: (i) a light chain of an anti-PD1 antibody, comprising, from N-terminus to C-terminus, the VL and CL domains of the anti-PD1 antibody, the VL and CL domains comprising or consisting of SEQ ID NOs: 16 and 18, respectively, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof;

[0740] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0741] In a very specific embodiment, the multifunctional molecule comprises or consists of:

[0742] - a first strand comprising or consisting of a sequence as shown in SEQ ID NO: 64, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof

[0743] - a second strand comprising or consisting of the sequence as shown in SEQ ID NO: 28, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof

[0744] - a third strand comprising or consisting of the sequence as shown in SEQ ID NO: 29, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, and

[0745] - a fourth strand comprising or consisting of the sequence as shown in SEQ ID NO: 20, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0746] In one embodiment, the multifunctional molecule comprises or consists in particular of:

[0747] a first chain which comprises or consists of, from N-terminus to C-terminus: (i) an anti-CD28 scFv comprising the VL and VH domains of an anti-CD28 antibody, preferably connected via a peptide linker, (ii) optionally a peptide linker, (iii) the VL and CH1 domains of an anti-PD1 antibody,

[0748] - a second chain, which comprises or consists of, from N-terminus to C-terminus: VH and CL of an anti-PD1 antibody, optionally a hinge, CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming a first Fc chain,

[0749] - a third chain, which comprises or consists of, from N-terminus to C-terminus: VH and CH1 of an anti-PD1 antibody, optionally a hinge, CH2 and CH3 domains of the antibody, said CH2 and CH3 domains forming a second Fc chain, and

[0750] - a fourth chain, which comprises or consists of (i) the VL and CL domains of an anti-PD1 antibody from N-terminus to C-terminus;

[0751] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0752] In particular, the first Fc chain is the "knob", and the second Fc chain is the "hole". Alternatively, the first Fc chain is the "hole", and the second Fc chain is the "knob".

[0753] Preferably, the multifunctional molecule comprises or consists of:

[0754] - a first chain comprising or consisting of: (i) an anti-CD28 scFv comprising a VL and a VH domain comprising or consisting of SEQ ID NOs: 45 and 44, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, preferably connected via a peptide linker, preferably SEQ ID NO: 57 (ii) optionally a peptide linker, preferably SEQ ID NO: 57, and (iii) a chain comprising, from N-terminus to C-terminus, the VL and CH1 domains of an anti-PD1 antibody comprising or consisting of SEQ ID NOs: 16 and 17, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof;

[0755] - a second chain, which comprises or consists of, from N-terminus to C-terminus: (i) VH and CL domains of an anti-PD1 antibody comprising or consisting of SEQ ID NOs: 15 and 18, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (ii) optionally a hinge, preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, and (iii) CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming a first Fc chain, which comprises or consists of SEQ ID NO: 25, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof;

[0756] - a third chain comprising or consisting of a heavy chain of an anti-PD1 antibody and comprising, from N-terminus to C-terminus, (i) a VH and CH1 domain of an anti-PD1 antibody comprising or consisting of SEQ ID NOs: 15 and 17, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (ii) optionally a hinge, preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, and (iii) CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming a second Fc chain comprising or consisting of SEQ ID NO: 26, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; and

[0757] - a fourth chain comprising or consisting of: (i) a light chain of an anti-PD1 antibody, comprising, from N-terminus to C-terminus, the VL and CL domains of an anti-PD1 antibody comprising or consisting of SEQ ID NOs: 16 and 18, respectively, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof;

[0758] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0759] In a very specific embodiment, the multifunctional molecule comprises or consists of:

[0760] - a first strand comprising or consisting of a sequence as shown in SEQ ID NO: 63, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof

[0761] - a second strand comprising or consisting of a sequence as shown in SEQ ID NO: 28, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof

[0762] - a third strand comprising or consisting of the sequence as shown in SEQ ID NO: 29, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, and

[0763] - a fourth strand comprising or consisting of the sequence as shown in SEQ ID NO: 20, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0764] In another embodiment, the multifunctional molecule comprises or consists in particular of:

[0765] - a first chain, which comprises or consists of the VL and CL domains of an anti-PD1 antibody from N-terminus to C-terminus;

[0766] - a second chain comprising, from N-terminus to C-terminus, the VH and CH1 domains of an anti-PD1 antibody, a hinge, the CH2 and CH3 domains of an antibody, or a combination thereof, the CH2 and CH3 domains forming a first Fc chain;

[0767] - a third chain, which comprises or consists of, from N-terminus to C-terminus: (i) the VH and CH1 domains of an anti-PD1 antibody, a hinge, the CH2 and CH3 domains of the antibody, the CH2 and CH3 domains forming a second Fc chain, optionally a peptide linker, and (iii) the VL and VH domains of an anti-CD28 antibody, preferably connected by a peptide linker; and

[0768] - a fourth chain, which comprises or consists of the VL and CL domains of an anti-PD1 antibody from N-terminus to C-terminus,

[0769] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0770] In particular, the first Fc chain is the "knob", and the second Fc chain is the "hole". Alternatively, the first Fc chain is the "hole", and the second Fc chain is the "knob".

[0771] Preferably, the multifunctional molecule comprises or consists of:

[0772] - a first chain, which is a light chain, which comprises or consists of the following from N-terminus to C-terminus: the VL and CL domains of an anti-PD1 antibody comprising or consisting of SEQ ID NOs: 16 and 18, respectively, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof;

[0773] - a second chain, which is a heavy chain, which comprises or consists of the following from N-terminus to C-terminus: (i) VH and CH1 domains of an anti-PD1 antibody comprising or consisting of SEQ ID NOs: 15 and 17, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (ii) optionally a hinge, preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof (iii) CH2 and CH3 domains of an antibody, which CH2 and CH3 domains form a first Fc chain, which comprises or consists of SEQ ID NO: 25, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof;

[0774] - A third chain, which comprises or consists of the following from N-terminus to C-terminus: a) a heavy chain, which comprises: (i) VH and CH1 domains of an anti-PD1 antibody comprising or consisting of SEQ ID NOs: 15 and 17, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (ii) optionally a hinge, preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, and (iii) CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming a second Fc chain, which comprises or consists of SEQ ID NO: 26, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (b) optionally a peptide linker, preferably SEQ ID NO: 57, and (c) an anti-CD28 scFv comprising the VL and VH domains of an anti-CD28 antibody, the VL and VH domains comprising SEQ ID NO: 45 and 44 or consisting thereof, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof, preferably connected via a peptide linker, preferably SEQ ID NO: 57, and

[0775] - a fourth chain, which is a light chain of an anti-PD1 antibody and comprises or consists of, from N-terminus to C-terminus: the VL and CL domains of an anti-PD1 antibody comprising or consisting of SEQ ID NOs: 16 and 18, respectively, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof;

[0776] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0777] In a very specific embodiment, the multifunctional molecule comprises or consists of:

[0778] - a first strand comprising or consisting of a sequence as shown in SEQ ID NO: 20, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof

[0779] - a second strand comprising or consisting of a sequence as shown in SEQ ID NO: 27, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof

[0780] - a third strand comprising or consisting of the sequence shown in SEQ ID NO: 62, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, and

[0781] - a fourth strand comprising or consisting of the sequence as shown in SEQ ID NO: 20, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0782] In another embodiment, the multifunctional molecule comprises or consists in particular of:

[0783] - A first chain comprising or consisting of the VL and CL domains of an anti-PD1 antibody from the N-terminus to the C-terminus

[0784] - a second chain, which comprises or consists of the VH and CH1 domains of an anti-PD1 antibody, a hinge, and the CH2 and CH3 domains of the antibody from the N-terminus to the C-terminus, the CH2 and CH3 domains forming the first Fc chain,

[0785] - a third chain, which comprises or consists of, from N-terminus to C-terminus: (i) the VH and CH1 domains of an anti-PD1 antibody, a hinge, the CH2 and CH3 domains of the antibody, the CH2 and CH3 domains forming a second Fc chain, optionally a peptide linker, and (iii) the VH and VL domains of an anti-CD28 antibody, preferably connected by a peptide linker;

[0786] - a fourth chain, which comprises or consists of the VL and CL domains of an anti-PD1 antibody from N-terminus to C-terminus,

[0787] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0788] In particular, the first Fc chain is the "knob", and the second Fc chain is the "hole". Alternatively, the first Fc chain is the "hole", and the second Fc chain is the "knob".

[0789] Preferably, the multifunctional molecule comprises or consists of:

[0790] - a first chain, which is a light chain of an anti-PD1 antibody and comprises or consists of, from N-terminus to C-terminus: the VL and CL domains of an anti-PD1 antibody comprising or consisting of SEQ ID NOs: 16 and 18, respectively, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof;

[0791] - a second chain, which is a heavy chain of an anti-PD1 antibody and comprises or consists of, from N-terminus to C-terminus: (i) VH and CH1 domains of an anti-PD1 antibody comprising or consisting of SEQ ID NOs: 15 and 17, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (ii) optionally a hinge, preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, and (iii) CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming a first Fc chain, which comprises or consists of SEQ ID NO: 25, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof;

[0792] - A third chain comprising or consisting of: a) a heavy chain comprising, from N-terminus to C-terminus, (i) VH and CH1 domains of an anti-PD1 antibody comprising or consisting of SEQ ID NOs: 15 and 17, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (ii) optionally a hinge, preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, (iii) CH2 and CH3 domains of an antibody, said CH2 and CH3 domains forming a second Fc chain, comprising or consisting of SEQ ID NO: 26, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (b) optionally a peptide linker, preferably SEQ ID NO: 57, and (c) an anti-CD28 scFv comprising the VH and VL domains of an anti-CD28 antibody, the VH and VL domains comprising, respectively, SEQ ID NO: 44 and 45 or consisting thereof, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof, preferably connected via a peptide linker, preferably SEQ ID NO: 57,

[0793] - a fourth chain, which is a light chain of an anti-PD1 antibody and comprises or consists of, from N-terminus to C-terminus: the VL and CL domains of an anti-PD1 antibody comprising or consisting of SEQ ID NOs: 16 and 18, respectively, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof;

[0794] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0795] In a very specific embodiment, the multifunctional molecule comprises or consists of:

[0796] - a first strand comprising or consisting of a sequence as shown in SEQ ID NO: 20, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof

[0797] - a second strand comprising or consisting of a sequence as shown in SEQ ID NO: 27, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof

[0798] - a third strand comprising or consisting of the sequence shown in SEQ ID NO: 61, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, and

[0799] - a fourth strand comprising or consisting of the sequence as shown in SEQ ID NO: 20, optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof.

[0800] Each of the above molecules comprises a single (one) anti-CD28 binding moiety.

[0801] Preferably, when mutations occur in VL or VH, one, two or three modifications are preferably located outside the CDRs (ie, in the framework regions).

[0802] In some embodiments, the anti-CD28 binding domain is a variant comprising one or more mutations in the CDR and / or framework regions. The anti-CD28 variants are specifically described in the above section "Second Binding Moiety". Any of the variants can be incorporated into the above exemplary multifunctional molecules.

[0803] In some aspects, the multifunctional molecule comprises or consists of:

[0804] - a first chain, which is a light chain of an anti-PD1 antibody and comprises or consists of, from N-terminus to C-terminus: a VL and CL domain comprising or consisting of SEQ ID NOs: 16 and 18, respectively, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof;

[0805] - a second chain, which is a heavy chain of an anti-PD1 antibody and comprises or consists of, from N-terminus to C-terminus: (i) VH and CH1 domains comprising or consisting of SEQ ID NOs: 15 and 17, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (ii) optionally a hinge, preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof (iii) CH2 and CH3 domains of an antibody, the CH2 and CH3 domains forming a first Fc chain, which comprises or consists of SEQ ID NO: 25, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; and

[0806] - A third chain, which comprises or consists of, from N-terminus to C-terminus: (i) an anti-CD28 scFv comprising:

[0807] (i)(a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 44, 83, 84, 85 and 88, optionally with one, two or three modifications selected from the group consisting of substitution, addition, deletion and any combination thereof; and

[0808] (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 86, 87, 89 and 90, optionally having one, two or three modifications selected from substitution, addition, deletion and any combination thereof; wherein VH and VL are preferably connected via a peptide linker, preferably SEQ ID NO: 57;

[0809] (ii) optionally a peptide linker, preferably SEQ ID NO: 57, (iii) optionally a hinge, preferably SEQ ID NO: 58, optionally having one, two or three modifications selected from substitution, addition, deletion and any combination thereof, iv) CH2 and CH3 domains of an antibody, the CH2 and CH3 domains forming a second Fc chain, comprising or consisting of SEQ ID NO: 26 or 54, preferably SEQ ID NO: 54, optionally having one, two or three modifications selected from substitution, addition, deletion and any combination thereof;

[0810] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0811] In some aspects, the multifunctional molecule comprises or consists of:

[0812] - a first chain, which is a light chain of an anti-PD1 antibody and comprises or consists of, from N-terminus to C-terminus: a VL and CL domain comprising or consisting of SEQ ID NOs: 16 and 18, respectively, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof;

[0813] - a second chain, which is a heavy chain of an anti-PD1 antibody and comprises or consists of, from N-terminus to C-terminus: (i) a VL and CH1 domain comprising or consisting of SEQ ID NOs: 15 and 17, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (ii) optionally a hinge, preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof (iii) CH2 and CH3 domains of an antibody, the CH2 and CH3 domains forming a first Fc chain, which comprises or consists of SEQ ID NO: 25, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; and

[0814] - A third chain, which comprises or consists of, from N-terminus to C-terminus: (i) an anti-CD28 scFv comprising:

[0815] (a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs 44, 83, 84, 85 and 88; and

[0816] (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 45, optionally having 1, 2 or 3 modifications selected from substitutions, additions, deletions and any combination thereof, preferably amino acid substitutions, wherein VH and VL are preferably connected via a peptide linker, preferably SEQ ID NO: 57;

[0817] (ii) optionally a peptide linker, preferably SEQ ID NO: 57, (iii) optionally a hinge, preferably SEQ ID NO: 58, optionally having one, two or three modifications selected from substitution, addition, deletion and any combination thereof, iv) CH2 and CH3 domains of an antibody, the CH2 and CH3 domains forming a second Fc chain, comprising or consisting of SEQ ID NO: 26 or 54, preferably SEQ ID NO: 54, optionally having one, two or three modifications selected from substitution, addition, deletion and any combination thereof;

[0818] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0819] In some aspects, the multifunctional molecule comprises or consists of:

[0820] - a first chain, which is a light chain of an anti-PD1 antibody and comprises or consists of, from N-terminus to C-terminus: a VL and CL domain comprising or consisting of SEQ ID NOs: 16 and 18, respectively, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof;

[0821] - a second chain, which is a heavy chain of an anti-PD1 antibody and comprises or consists of, from N-terminus to C-terminus: (i) a VL and CH1 domain comprising or consisting of SEQ ID NOs: 15 and 17, respectively, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; (ii) optionally a hinge, preferably SEQ ID NO: 58, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof (iii) CH2 and CH3 domains of an antibody, the CH2 and CH3 domains forming a first Fc chain, which comprises or consists of SEQ ID NO: 25, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; and

[0822] - A third chain, which comprises or consists of, from N-terminus to C-terminus: (i) an anti-CD28 scFv comprising:

[0823] (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 44, optionally having 1, 2 or 3 modifications selected from substitutions, additions, deletions and any combination thereof, preferably amino acid substitutions; and

[0824] (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 86, 87, 89 and 90, wherein VH and VL are preferably connected via a peptide linker, preferably SEQ ID NO: 57;

[0825] (ii) optionally a peptide linker, preferably SEQ ID NO: 57, (iii) optionally a hinge, preferably SEQ ID NO: 58, optionally having one, two or three modifications selected from substitution, addition, deletion and any combination thereof, iv) CH2 and CH3 domains of an antibody, the CH2 and CH3 domains forming a second Fc chain, comprising or consisting of SEQ ID NO: 26 or 54, preferably SEQ ID NO: 54, optionally having one, two or three modifications selected from substitution, addition, deletion and any combination thereof;

[0826] Wherein the first and second Fc chains are complementary and thereby form an Fc domain.

[0827] Preparation of multifunctional molecules - nucleic acid molecules encoding the multifunctional molecules of the present invention, recombinant expression vectors and Host cells containing these nucleic acid molecules and recombinant expression vectors

[0828] In order to produce the multifunctional molecule according to the present invention, in particular by mammalian cell production, the nucleotide sequence or nucleotide sequence group encoding the multifunctional molecule is subcloned into one or more expression vectors. Such vectors are generally used for transfecting mammalian cells. The general technique of producing molecules comprising antibody sequences is described in Coligan et al. (eds.), Current protocols in immunology, at pp. 10.19.1-10.19.11 (Wiley Interscience 1992), the contents of which are incorporated herein by reference, and described in "Antibody engineering: a practical guide" from WH Freeman and Company (1992), wherein the comments related to the generation of molecules are scattered in the corresponding text.

[0829] Typically, such methods include the following steps:

[0830] (1) transfecting or transforming a suitable host cell with a polynucleotide encoding the recombinant multifunctional molecule of the present invention or a vector containing the polynucleotide;

[0831] (2) culturing the host cell in a suitable culture medium; and

[0832] (3) optionally isolating and / or purifying the multifunctional molecule from the culture medium or the host cells.

[0833] The present invention further relates to nucleic acids encoding the above-mentioned multifunctional molecules, vectors comprising the nucleic acids of the present invention, preferably expression vectors, genetically engineered host cells transformed with the vectors of the present invention or directly with sequences encoding recombinant multifunctional molecules, and methods for producing the multifunctional molecules of the present invention by recombinant technology.

[0834] Nucleic acids, vectors and host cells are described in more detail below.

[0835] Nucleic acid sequence

[0836] The present invention further relates to nucleic acid molecules encoding multifunctional molecules as defined above or nucleic acid molecule groups encoding multifunctional molecules as defined above. Nucleic acids encoding multifunctional molecules disclosed herein can be amplified by any technology known in the art, such as PCR. Such nucleic acids can be easily separated and sequenced using conventional procedures known to those skilled in the art.

[0837] Preferably, the present invention relates to an isolated nucleic acid molecule encoding a multifunctional molecule as defined above or a set of isolated nucleic acid molecules encoding a multifunctional molecule as defined above.

[0838] In particular aspects, a nucleic acid molecule encoding a multifunctional molecule as defined herein comprises or consists of a nucleic acid molecule or a group of nucleic acid molecules encoding different chains of the multifunctional molecules described herein (particularly in the section "Examples of multifunctional molecules").

[0839] In one embodiment, the nucleic acid molecule is an isolated, particularly a non-natural nucleic acid molecule.

[0840] Carrier

[0841] In another aspect, the present invention relates to a vector comprising a nucleic acid molecule or a group of nucleic acid molecules as defined above.

[0842] As used herein, "vector" is a nucleic acid molecule used as a vehicle to transfer genetic material into a cell. The term "vector" encompasses plasmids, viruses, cosmids, and artificial chromosomes. Typically, engineered vectors include an origin of replication, a multiple cloning site, and a selectable marker. The vector itself is typically a nucleotide sequence, typically a DNA sequence, which includes an insert (transgene) and a larger sequence used as a vector "backbone." Modern vectors can encompass other features except transgenic inserts and backbones: promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, protein purification tags. Vectors referred to as expression vectors (expression constructs) are specifically used to express transgenes in target cells and typically have control sequences.

[0843] Those skilled in the art can clone the nucleic acid molecule or nucleic acid molecule group encoding the multifunctional molecule into a vector and then transform it into a host cell. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. Methods known to those skilled in the art can be used to construct an expression vector containing the nucleic acid sequence of the multifunctional molecule, the variant described herein, and the appropriate regulatory components for transcription / translation.

[0844] Therefore, the present invention also provides a recombinant vector comprising a nucleic acid molecule or a nucleic acid molecule group encoding a multifunctional molecule according to the present invention. In a preferred aspect, the expression vector further comprises a promoter and a nucleic acid sequence encoding a secretion signal peptide, and optionally at least one drug resistance gene for screening. The expression vector may further comprise a ribosome binding site, a transcription terminator, etc. for initiating translation.

[0845] Expression vectors can be introduced into host cells using a variety of techniques, including calcium phosphate transfection, liposome-mediated transfection, electroporation, etc. Preferably, transfected cells are selected and expanded, wherein the expression vector is stably integrated into the host cell genome to produce stable transformants.

[0846] Host cells

[0847] In another aspect, the invention relates to a host cell comprising a vector or a nucleic acid molecule or a set of nucleic acid molecules as defined above, eg for the purpose of multifunctional molecule production.

[0848] As used herein, the term "host cell" is intended to include any single cell or cell culture, which can be or has been a receptor for a vector, an exogenous nucleic acid molecule, and a polynucleotide encoding a multifunctional molecule according to the present invention. The term "host cell" is also intended to include the progeny or potential progeny of a unicellular cell. Suitable host cells include prokaryotic cells or eukaryotic cells, and also include, but are not limited to, bacteria, yeast cells, fungal cells, plant cells, and animal cells, such as insect cells and mammalian cells, such as mice, rats, rabbits, macaques, or humans.

[0849] Suitable host cells are in particular eukaryotic host cells that provide suitable post-translational modifications, such as glycosylation. Preferably, such suitable eukaryotic host cells may be fungi, such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe; insect cells, such as Mythimna separate; plant cells, such as tobacco, and mammalian cells, such as BHK cells, 293 cells, CHO cells, NSO cells and COS cells.

[0850] Preferably, the host cell of the present invention is selected from the group consisting of CHO cells, COS cells, NSO cells and HEK cells.

[0851] The host cell then stably or transiently expresses the multifunctional molecule according to the invention. Such expression methods are known to those skilled in the art.

[0852] Also disclosed herein is a method for producing the multifunctional molecule. The method includes, under conditions suitable for its expression, culturing a host cell comprising a nucleic acid or a nucleic acid group encoding a multifunctional molecule as provided above, and optionally recovering the multifunctional molecule from the host cell (or host cell culture medium). In particular, for the recombinant production of multifunctional molecules, for example, as described above, nucleic acids or nucleic acid groups encoding multifunctional molecules are separated and inserted into one or more vectors for further cloning and / or expression in host cells. Then, the multifunctional molecule is separated and / or purified by any method known in the art. These methods include, but are not limited to, conventional renaturation treatment, treatment by a protein precipitant (e.g., salt precipitation), centrifugation, cell lysis by penetration, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography or gel chromatography, adsorption chromatography, ion exchange chromatography, HPLC, any other liquid chromatography and combinations thereof. Multifunctional molecule separation techniques particularly include affinity chromatography, size exclusion chromatography and ion exchange chromatography using protein-A Sepharose. Protein A is preferably used to separate the multifunctional molecule of the present invention.

[0853] Methods for selecting suitable multifunctional molecules

[0854] In one aspect, the present invention relates to a method for selecting a multifunctional molecule of the present invention, the method comprising or consisting of at least one of the following steps:

[0855] a. selecting a first binding moiety that binds to a target specifically expressed on the surface of an immune cell; and wherein the target specifically expressed on the surface of an immune cell is preferably selected from the group consisting of PD-1, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, TIM3, CD39, CD30, NKG2D, NKG2A, LAG3, 2B4, DR3, CD101, CD44, CD38, CXCR3, CXCR5, CD4, CD8, CD25, CRTAM, CD96, CD226, CD112R, CD103, CEACAM and CD122, preferably selected from the group consisting of PD-1, VISTA, CTLA-4, BTLA, TIGIT, CD160, LAG3 and TIM3, and preferably PD-1

[0856] b. Select the structure of the multifunctional molecule;

[0857] c. constructing / cloning a multifunctional molecule comprising an anti-CD28 binding domain and a first binding moiety;

[0858] d. Testing the ability of the multifunctional molecule to bind to: PD-1, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, TIM3, CD39, CD30, NKG2D, NKG2A, LAG3, 2B4, DR3, CD101, CD44, CD38, CXCR3, CXCR5, CD4, CD8, CD25, CRTAM, CD96, CD226, CD112R, CD103, CEACAM and CD122, preferably selected from the group consisting of PD-1, VISTA, CTLA-4, BTLA, TIGIT, CD160, LAG3 and TIM3, more preferably the ability to bind to PD-1, and optionally the ability of the multifunctional molecule to inhibit the binding of human PD-L1 and / or PD-L2 to human PD-1;

[0859] e. Testing the ability of the multifunctional molecule to bind to CD28 and the ability of the multifunctional molecule to have agonist activity on CD28;

[0860] f. Testing:

[0861] ii) the ability of the multifunctional molecule to bind in cis to immune cells, in particular T cells; and / or

[0862] iii) the ability of the multifunctional molecule not to exhibit trans-activity, i.e., the ability to co-target tumor cells and T cells;

[0863] and optionally comprising the following steps

[0864] g. Select a multifunctional molecule that specifically binds to a target specifically expressed on the surface of an immune cell, the target preferably being selected from the group consisting of PD-1, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, TIM3, CD39, CD30, NKG2D, NKG2A, LAG3, 2B4, DR3, CD101, CD44, CD38, CX CR3, CXCR5, CD4, CD8, CD25, CRTAM, CD96, CD226, CD112R, CD103, CEACAM and CD122, preferably selected from the group consisting of PD-1, VISTA, CTLA-4, BTLA, TIGIT, CD160, LAG3 and TIM3, more preferably PD1; and a multifunctional molecule that specifically binds to CD28 and can cis-bind to immune cells, especially T cells, and / or does not exhibit trans activity.

[0865] The method for selecting the multifunctional molecule of the present invention can advantageously be further performed according to the method for producing a multifunctional molecule of the present invention as described above.

[0866] Pharmaceutical composition and method of administration thereof

[0867] The present invention further relates to pharmaceutical compositions, which comprise multifunctional molecules as described herein, nucleic acid molecules, nucleic acid molecule groups, carriers and / or host cells as described above, preferably as active ingredients or compounds. The preparations can be sterilized, and if necessary, can be mixed with adjuvants, for example, pharmaceutically acceptable carriers, excipients, salts, antioxidants and / or stabilizers, which do not interact harmfully with the multifunctional molecules of the present invention, nucleic acids, carriers and / or host cells of the present invention, and do not impart any undesirable toxicological effects. Optionally, the pharmaceutical composition can further comprise other therapeutic agents.

[0868] In particular, the pharmaceutical composition according to the present invention can be formulated for any conventional route of administration, including topical, enteral, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration, etc. For ease of administration, the multifunctional molecules described herein can be prepared into pharmaceutical compositions for in vivo administration. Such methods of such compositions have been described in the art (see, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st edition (2005).

[0869] Pharmaceutical compositions can be prepared by mixing the multifunctional molecule with the desired purity with optional pharmaceutically acceptable carriers, excipients, antioxidants and / or stabilizers in the form of lyophilized formulations or aqueous solutions. Such suitable carriers, excipients, antioxidants and / or stabilizers are well known in the art and have been described in, for example, Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0870] To facilitate delivery, any multifunctional molecule or its encoding nucleic acid can be conjugated to a chaperone. The chaperone can be a naturally occurring substance, such as a protein (e.g., human serum albumin, low-density lipoprotein or globulin), a carbohydrate (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid) or a lipid. It can also be a recombinant or synthetic molecule, e.g., a synthetic polymer, e.g., a synthetic polypeptide.

[0871] The pharmaceutical composition according to the present invention can be formulated to release the active ingredient (e.g., the multifunctional molecule of the present invention) substantially immediately after administration or at any predetermined time or time period after administration. In some aspects, the pharmaceutical composition can adopt a timed release, delayed release, and sustained release delivery system so that the delivery of the composition occurs before the sensitization of the part to be treated, and there is enough time to cause the sensitization of the part to be treated. Methods known in the art can be used to prevent or minimize the release and absorption of the composition until it reaches the target tissue or organ, or to ensure the timed release of the composition.

[0872] Those skilled in the art will appreciate that the preparations of the present invention can be isotonic with human blood, i.e., the preparations of the present invention have substantially the same osmotic pressure as human blood. Such isotonic preparations typically have an osmotic pressure of about 250 mOSm to about 350 mOSm. Isotonicity can be measured, for example, by vapor pressure or a frozen osmometer.

[0873] Pharmaceutical compositions generally must be sterile and stable under the conditions of manufacture and storage. Prevention of the presence of microorganisms can be ensured by sterilization procedures (e.g., by microfiltration) and / or by the inclusion of various antibacterial and antifungal agents.

[0874] The amount of active ingredients that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the specific mode of administration. The amount of active ingredients that can be combined with a carrier material to produce a single dosage form is generally the amount of the composition that produces a therapeutic effect. Preferably, the pharmaceutical compositions described herein are generally administered in an amount and time required or sufficient to induce an immune response.

[0875] Use in treating diseases

[0876] The multifunctional molecules, nucleic acids, vectors, host cells, compositions and methods of the present invention have many uses and applications in vitro and in vivo. In particular, any multifunctional molecule, nucleic acid molecule, nucleic acid molecule group, vector, host cell or pharmaceutical composition provided herein can be used for therapeutic methods and / or therapeutic purposes.

[0877] The present invention also relates to multifunctional molecules, nucleic acids or vectors encoding such multifunctional molecules, or pharmaceutical compositions comprising such multifunctional molecules or nucleic acids or vectors, for use in treating a subject's condition and / or disease and / or as a medicament or vaccine. It also relates to the use of multifunctional molecules as described herein; nucleic acids or vectors encoding such multifunctional molecules, or pharmaceutical compositions comprising such multifunctional molecules or nucleic acids or vectors in treating a subject's disease and / or condition. It also relates to the use of multifunctional molecules as described herein; nucleic acids or vectors encoding such multifunctional molecules, or pharmaceutical compositions comprising such multifunctional molecules or nucleic acids or vectors in the preparation of a medicament for treating a subject's disease and / or condition. Finally, it relates to a method for treating a disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition or a multifunctional molecule, or a nucleic acid or vector encoding such a nucleic acid.

[0878] The use of the multifunctional molecule, nucleic acid, nucleic acid set, vector encoding the multifunctional molecule or pharmaceutical composition comprising the multifunctional molecule or nucleic acid or nucleic acid set or vector according to the present invention has application in all T lymphocyte-dependent pathological conditions.

[0879] Such a pathological condition may be, for example, cancer or an infectious disease.

[0880] In one aspect, the present invention relates to a method for treating a disease and / or condition in a subject in need thereof, the disease and / or condition being selected from the group consisting of cancer and infectious diseases, the method comprising administering to the subject an effective amount of a multifunctional molecule or pharmaceutical composition as defined above. Examples of such diseases are described in more detail below.

[0881] In one aspect, the method of treatment comprises: (a) identifying a patient in need of treatment; and (b) administering to the patient a therapeutically effective amount of a multifunctional molecule, nucleic acid, set of nucleic acids, vector or pharmaceutical composition as described herein.

[0882] A subject in need of treatment may be a person who has a disease, is at risk of having a disease, or is suspected of having a disease. Such patients can be identified by routine physical examination.

[0883] In another aspect, the multifunctional molecules disclosed herein can be administered to a subject, e.g., in vivo, to enhance immunity, preferably to treat a condition and / or disease. Accordingly, in one aspect, the invention provides a method for altering an immune response in a subject, comprising administering to the subject a multifunctional molecule, nucleic acid, vector or pharmaceutical composition of the invention, thereby altering the immune response in the subject. Preferably, the immune response is enhanced, improved, stimulated or upregulated.

[0884] cancer

[0885] In another aspect, the present invention provides the use of a multifunctional molecule or pharmaceutical composition as disclosed herein in the preparation of a medicament for treating cancer, such as for inhibiting the growth of tumor cells in a subject.

[0886] The present invention also provides a multifunctional molecule, composition or pharmaceutical composition for use in treating a subject suffering from cancer.

[0887] The present invention also provides a method for treating cancer in a subject, comprising administering a therapeutically effective amount of a pharmaceutical composition, composition or multifunctional molecule as disclosed herein.

[0888] As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the blood and lymphatic system.

[0889] Therefore, in one aspect, the present invention provides a method for treating cancer, such as a method for inhibiting the growth of tumor cells in a subject, the method comprising administering to the subject a therapeutically effective amount of a multifunctional molecule or pharmaceutical composition according to the present invention. In particular, the present invention relates to treating a subject using a multifunctional molecule to inhibit the growth of cancer cells.

[0890] Any suitable cancer that can be treated with the methods provided herein can be a hematopoietic cancer or a solid cancer. Such cancers include carcinoma, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, head and neck cancer, renal cancer, liver cancer, lung cancer, lymphoma, glioma, mesothelioma, melanoma, gastric cancer, urethral cancer, environmentally induced cancers and any combination of the cancers. In addition, the present invention includes refractory or recurrent malignancies. Preferably, the cancer to be treated or prevented is selected from the group consisting of metastatic or non-metastatic melanoma, malignant mesothelioma, non-small cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck cancer, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, small cell lung cancer metastatic Merkel cell carcinoma, gastric cancer or gastroesophageal cancer and cervical cancer.

[0891] In a particular aspect, the cancer is a hematological malignancy or a solid tumor. Such cancer may be selected from the group consisting of hematolymphoid neoplasm, angioimmunoblastic T-cell lymphoma, myelodysplastic syndrome, acute myeloid leukemia.

[0892] In a particular aspect, cancer is a cancer induced by a virus or associated with immunodeficiency. Such cancers can be selected from the group consisting of Kaposi's sarcoma (e.g., associated with Kaposi's sarcoma herpesvirus), squamous cell carcinomas of the cervix, anus, penis and vulva, and oropharyngeal cancer (e.g., associated with human papillomavirus), B-cell non-Hodgkin's lymphoma (NHL), including diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmablastic lymphoma, primary central nervous system lymphoma, HHV-8 primary effusion lymphoma, classical Hodgkin's lymphoma, and lymphoproliferative diseases (e.g., associated with Epstein-Barr virus (EBV) and / or Kaposi's sarcoma herpesvirus), hepatocellular carcinoma (e.g., associated with hepatitis B and / or C virus), Merkel cell carcinoma (e.g., associated with Merkel cell polyomavirus (MPV)), and cancers associated with human immunodeficiency virus (HIV) infection.

[0893] Preferred cancers for treatment include cancers that are generally responsive to immunotherapy. Alternatively, preferred cancers for treatment are cancers that are not responsive to immunotherapy.

[0894] Infectious Diseases

[0895] The multifunctional molecules, nucleic acids, nucleic acid groups, vectors, host cells or pharmaceutical compositions of the present invention can be used to treat patients exposed to specific toxins or pathogens. Accordingly, one aspect of the present invention provides a method for treating an infectious disease in a subject, the method comprising administering to the subject a multifunctional molecule according to the present invention or a pharmaceutical composition comprising such a multifunctional molecule, preferably, such that the infectious disease in the subject is treated.

[0896] Any suitable infection can be treated using the multifunctional molecules, nucleic acids, sets of nucleic acids, vectors, host cells or pharmaceutical compositions provided herein.

[0897] Some examples of pathogenic viruses that cause infection that can be treated by the methods of the present invention include: human immunodeficiency virus (HIV), hepatitis (type A, B, or C), herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV-II and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, human T-lymphotropic virus (HTLV), dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus.

[0898] Some examples of pathogenic bacteria causing infections that can be treated by the methods of the present invention include: Chlamydia, Rickettsia, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Meningococcus and Gonorrhea, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Botulinum, Anthrax, Plague, Leptospirosis, and Lyme disease bacteria.

[0899] Pathogenic fungi causing infections that can be treated by the methods of the present invention include Candida (Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, etc.), Cryptococcus neoformans, Aspergillus (Aspergillus fumigatus, Aspergillus niger, etc.), genera of the order Mucorales (Mucor, Absidia, Rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0900] Pathogenic parasites that cause infections that can be treated by the methods of the present invention include: Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lamblia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondi, and Nippostrongylus brasiliensis.

[0901] Combination or combination therapy

[0902] In one aspect, the invention relates to a combination of a) a multifunctional molecule of the invention and b) a different therapeutic agent, preferably an anticancer agent or an anti-infective agent. Such a combination may in particular be comprised in a pharmaceutical composition as described herein.

[0903] The multifunctional molecules according to the present invention can be combined with other potential strategies to overcome the immune escape mechanisms of drugs in clinical development or on the market (see, for example, Table 1 in Antonia et al. Immuno-oncology combinations: a review of clinical experience and future prospects. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 20, 6258–6268, 2014). Such combinations with multifunctional molecules according to the present invention are particularly suitable for:

[0904] 1-Reversing the suppression of adaptive immunity (blocking T cell checkpoint pathways);

[0905] 2- Initiation of adaptive immunity (using agonist molecules, especially antibodies, to promote T cell co-stimulatory receptor signaling),

[0906] 3- Improve the function of innate immune cells;

[0907] 4- Activation of the immune system (enhancing immune cell effector functions), for example through vaccine-based strategies.

[0908] Therefore, the present invention also provides a combination therapy with any multifunctional molecule as described herein or a pharmaceutical composition comprising such multifunctional molecules and a suitable second agent for treating a disease or condition. In one aspect, as described above, the multifunctional molecule and the second agent may be present in a separate (or single) pharmaceutical composition. Alternatively, as used herein, the term "combination therapy" or "combined therapy" includes administering the two agents (e.g., multifunctional molecules as described herein and another or second suitable therapeutic agent) in a sequential manner, that is, wherein each therapeutic agent is administered at different times, and administering these therapeutic agents or at least two agents in a substantially simultaneous manner. Each agent may be administered sequentially or substantially simultaneously and may be affected by any appropriate route. The agent may be administered by the same or different routes. For example, the first agent (e.g., a multifunctional molecule) may be administered orally, and another therapeutic agent (e.g., an anticancer agent, an anti-infective agent, or an immunomodulator) may be administered intravenously. Alternatively, the agent selected in the combination may be administered by intravenous injection, while the other agents in the combination may be administered orally.

[0909] In one aspect, the additional therapeutic agent can be selected from a non-exhaustive list comprising: alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotic agents, antiproliferative agents, antiviral agents, Aurora kinase inhibitors, apoptosis promoters (e.g., Bcl-2 family inhibitors), death receptor pathway activators, Bcr-Abl kinase inhibitors, BiTE (bispecific T cell engager) antibodies, antibody drug conjugates, biological response modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, leukemia viral oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormone therapy, apoptosis protein (IAP) inhibitors, intercalating antibiotics ( intercalatingantibiotic), kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNA, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, nonsteroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate) ribose polymerase (PARP) inhibitors, platinum chemotherapy agents, polo-like (Plk) kinase inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoids, plant alkaloids, small inhibitory ribonucleic acids (siRNA), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, peptide vaccines, etc., epitopes or new epitopes from tumor antigens, and combinations of one or more of these agents.

[0910] For example, the additional therapeutic agent can be selected from the group consisting of chemotherapy, radiotherapy, targeted therapy, anti-angiogenic agents, hypomethylating agents, cancer vaccines, epitopes or neo-epitopes from tumor antigens, myeloid checkpoint inhibitors, other immunotherapies, and HDAC inhibitors.

[0911] In one aspect, the present invention relates to a combination therapy as defined above, wherein the second therapeutic agent is particularly selected from the group consisting of therapeutic vaccines, immune checkpoint blockers or activators, particularly adaptive immune cells (T and B lymphocytes) and antibody drug conjugates. Preferably, suitable agents for use with any multifunctional molecule according to the present invention or used in conjunction with a pharmaceutical composition include antibodies bound to co-stimulatory receptors (e.g., OX40, CD40, ICOS, CD27, HVEM or GITR), agents for inducing immunogenic cell death (e.g., chemotherapeutic agents, radiotherapeutic agents, anti-angiogenic agents or agents for targeted therapy), agents for inhibiting checkpoint molecules (e.g., CTLA4, LAG3, TIM3, BTLA or TIGIT), cancer vaccines, agents for modifying immunosuppressive enzymes (e.g., IDO1 or iNOS), agents for targeting Treg cells, agents for adoptive cell therapy or agents for regulating myeloid cells.

[0912] In one aspect, the invention relates to a combination therapy as defined above, wherein the second therapeutic agent is an immune checkpoint blocker or an adaptive immune cell (T and B lymphocyte) activator selected from anti-CTLA4, anti-CD2, anti-CD40, anti-HVEM, anti-BTLA, anti-CD160, anti-TIGIT, anti-TIM-1 / 3, anti-LAG-3, anti-2B4 and anti-OX40, anti-CD40 agonists, CD40-L, TLR agonists, anti-ICOS, ICOS-L and B-cell receptor agonists.

[0913] The present invention also relates to a method for treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of a multifunctional molecule or pharmaceutical composition as described herein, and a therapeutically effective amount of an additional or second therapeutic agent.

[0914] Specific examples of additional or second therapeutic agents are provided in WO 2018 / 053106, pages 36-43.

[0915] In a preferred aspect, the second therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a radiotherapeutic agent, an immunotherapeutic agent, a cell therapy agent (such as CAR-T cells), an antibiotic, and a probiotic.

[0916] Combination therapy may also rely on the administration of a multifunctional molecule according to the invention or a pharmaceutical composition comprising such a molecule in combination with surgery.

[0917] In particular, the multifunctional molecule according to the present invention is used in combination with a second multifunctional molecule, which second multifunctional molecule comprises at least one antigen binding domain, which antigen binding domain binds to a target specifically expressed on the surface of an immune cell that is different from that bound by the first multifunctional molecule, and / or the multifunctional molecule according to the present invention is used in combination with a multifunctional molecule comprising at least one immunostimulatory cytokine.

[0918] The multifunctional molecule of the present invention and the second multifunctional molecule can be administered simultaneously or sequentially. In the example of sequential administration, the multifunctional molecule of the present invention is administered before the second multifunctional molecule is administered. In another example, the multifunctional molecule of the present invention is administered after the second multifunctional molecule is administered.

[0919] Subjects, Protocol, and Administration

[0920] The present invention relates to a multifunctional molecule, nucleic acid, nucleic acid group or vector encoding such nucleic acid or nucleic acid group, host cell or pharmaceutical composition as disclosed herein, which is used as a drug or for treating a disease or for administration in a subject. It also relates to a method for treating a disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition or a multifunctional molecule.

[0921] The subject to be treated may be a human, in particular a human in the prenatal stage, a newborn, a child, an infant, a teenager or an adult, in particular an adult of at least 20, 30, 40 years old, preferably an adult of at least 50 years old, more preferably an adult of at least 60 years old.

[0922] In particular aspects, the subject can be immunosuppressed or immunocompromised. An immunocompromised subject refers to a subject who is usually unable to develop or is unlikely to develop a strong immune response due to disease, malnutrition or immunosuppressive therapy. People who may be considered immunosuppressed or immunocompromised include, but are not limited to, subjects who have received immunosuppressant therapy or are candidates for immunosuppressant therapy, subjects with AIDS (or HIV positive), subjects with severe combined immunodeficiency, diabetics, subjects who have received transplant surgery and are receiving immunosuppressants, and subjects who are receiving cancer chemotherapy. Immunosuppressed individuals also include subjects with most types of cancer (except skin cancer), sickle cell anemia, cystic fibrosis, asplenia, end-stage renal disease (dialysis), and subjects who have frequently received corticosteroids or other immunosuppressive therapies in the past year.

[0923] In some specific embodiments, the subject suffers from a disease involving the PD-1 / PDL-1 pathway, in particular wherein at least one of the PD-1 ligands (such as PDL-1 and / or PDL-2) or PD-1 is expressed, in particular overexpressed. Preferably, the subject suffers from cancer, even more preferably, from a PD1, PD-L1 and / or PD-L2 positive cancer or a PD-1 positive cancer.

[0924] In a specific embodiment, the subject has received at least one line of treatment, preferably multiple lines of treatment, prior to the administration of the multifunctional molecule according to the invention or the pharmaceutical composition according to the invention.

[0925] In certain embodiments, the patient is resistant to therapy, particularly anti-cancer therapy, particularly immune checkpoint inhibitors such as anti-PD-1 immunotherapy.

[0926] Conventional methods known to those of ordinary skill in the medical field can be used to administer the multifunctional molecules or pharmaceutical compositions disclosed herein to a subject, depending on the type of disease or disease site to be treated, e.g., orally, parenterally, enterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. Preferably, the multifunctional molecules or pharmaceutical compositions are administered via subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intratumoral, intrasternal, intrathecal, intralesional, and intracranial or infusion techniques.

[0927] The form of the pharmaceutical composition according to the present invention, the route of administration and the dosage of the pharmaceutical composition or the multifunctional molecule can be adjusted by those skilled in the art according to the type and severity of the infection and according to the patient, especially the age, weight, body shape, sex and / or general physical condition of the patient. The composition of the present invention can be applied in a variety of ways depending on whether local or systemic treatment is needed.

[0928] Reagent test kit

[0929] Any multifunctional molecule or composition described herein can be included in the kits provided herein. The present disclosure particularly provides kits for treating a disease or disorder (eg, cancer and / or infection).

[0930] In the context of the present invention, the term "kit" refers to two or more components (one of which corresponds to the multifunctional molecule, nucleic acid molecule, vector or cell of the present invention) packaged in a container, receptacle or other device. Thus, a kit can be described as a set of products and / or appliances sufficient to achieve a certain goal, which can be sold as a single unit. The kit of the present invention is in suitable packaging.

[0931] In particular, the kit according to the invention may comprise:

[0932] - a multifunctional molecule as defined above,

[0933] - a pharmaceutical composition comprising said multifunctional molecule,

[0934] - a nucleic acid molecule or a group of nucleic acid molecules encoding the multifunctional molecule,

[0935] - a vector comprising said nucleic acid molecule or nucleic acid molecule group, and / or

[0936] - a cell comprising said vector or nucleic acid molecule or group of nucleic acid molecules.

[0937] The kit may include the pharmaceutical composition or multifunctional molecule of the invention, and / or a host cell, and / or a vector encoding a nucleic acid of the invention in a suitable container.

[0938] The components, in particular the pharmaceutical compositions, comprised in the kit according to the invention may in particular be formulated as syringe-compatible compositions.

[0939] In certain embodiments, the kit further includes additional agents for treating cancer or infectious diseases, and the additional agents may be combined with the pharmaceutical composition, fusion protein or multifunctional molecule and / or host cell of the present invention, and / or a vector encoding the nucleic acid molecule of the present invention, and / or a nucleic acid molecule or other components of the kit of the present invention, or may be provided separately in the kit. In particular, the kit described herein may include one or more additional therapeutic agents, such as those described in the above-mentioned "combination therapy". The kit may be tailored to a specific cancer of an individual and include a corresponding second cancer therapy for the individual as described above.

[0940] Instructions for use of the multifunctional molecules or pharmaceutical compositions described herein typically include information such as dosage, time of administration, route of administration for the intended treatment, methods for reconstituting the multifunctional molecules of the invention, and / or methods for diluting the multifunctional molecules of the invention.

[0941] The instructions provided in the kits of the invention are typically written instructions on a label or package insert (eg, a piece of paper included in the kit in the form of a leaflet or instruction sheet). BRIEF DESCRIPTION OF THE DRAWINGS

[0942] Figure 1 : Example of designing an anti-PD-1 / anti-CD28 multifunctional molecular configuration with optimal cis-efficacy.

[0943] Figure 2 :Anti-PD-1*1 / anti-CD28*1 configuration B1 showed effective binding to human PD-1 and antagonistic activity by blocking PD-L1 / PD-1 interaction. (A) PD-1 binding ELISA assay. Human recombinant PD-1 (rPD1) protein was immobilized and different concentrations of anti-PD-1*1 / anti-CD28*1 configuration B1 were added. Revealed with anti-human Fc antibody coupled to peroxidase. Colorimetric method was performed at 450 nm using TMB substrate. (B) PD-1 binding cell counting assay. Anti-PD-1*1 / anti-CD28*1 configuration B1 was incubated with human PD-1 transduced CHO at 100 nM. Revealed with PE-labeled anti-human Fc antibody. Binding was determined by measuring the MFI (mean fluorescence intensity) of the PE signal by flow cytometry. (C) Antagonist PD-1 / PD-L1 ELISA assay. Human recombinant PD-L1 (rPDL1) protein was immobilized, and different concentrations of anti-PD-1*1 / anti-CD28*1 configuration B1 were mixed with a fixed concentration of biotinylated human PD-1 and added to the immobilized PD-L1. It was revealed with streptavidin coupled to peroxidase. The colorimetric method was performed at 450 nm using TMB substrate.

[0944] Figure 3 :Anti-PD-1*1 / anti-CD28*1 Configuration B1 binds effectively to human CD-28. CD28 binding MSD assay. Biotinylated human recombinant CD28 protein was immobilized on a streptavidin plate for MSD, and different concentrations of anti-PD-1*1 / anti-CD28*1 scFv Configuration B1 were added. Revealed with anti-human Fc antibody coupled to SulfoTag. The electrical signal was measured using the MSD system.

[0945] Figure 4 : The cis-activity of anti-PD-1*1 / anti-CD28 configuration B1 specifically reactivates PD-1+ CD28+ T cells and leaves PD-1neg T cells. (A) Schematic diagram of cis-activation of anti-PD-1 / CD28 bispecific antibodies. (B) IL-2 secretion bioassay of PD-1+CD28+ or PD-1-CD28+ Jurkat cells after treatment with anti-PD-1 / anti-CD28 multifunctional molecules. Briefly, PD-1+ CD28+ Jurkat cell lines were incubated on CD3-coated plates (2,5 ug / mL) in the presence of PD-1*1 / anti-CD28*1 scFv B1 configuration (100 nM), anti-PD-1*1, isotype control or anti-CD28 (bivalent anti-CD28 positive control for nonspecific PD-1-independent activation). After 48 hours of stimulation, IL-2 secretion in the culture supernatant was quantified. N=5 independent experiments. IL-2 secretion was normalized to the no antibody control condition. (C) IL-2 dose response, using anti-PD-1*1 CD28*1 configuration B1 ( ) or negative control treated PD-1+CD28+ Jurkat T cells: isotype control ( ) or anti-PD-1*1( ). One representative experiment out of 5 independent experiments.

[0946] Figure 5 : Anti-PD-1*1 / anti-CD28 configuration B1 has no trans-activity against PD-1-negative CD28+ T cells. (A) Schematic diagram of the experimental setup, PD-1 recombinant protein was coated and PD-1-CD28+ jurkat T cells were incubated with 100 nM of PD-1*1 / anti-CD28*1 scFv configuration B1 (100 nM), anti-PD-1*1, isotype control or anti-CD28 (bivalent anti-CD28 positive control for non-specific PD-1-independent activation). IL-2 secretion in the supernatant was quantified after 48 h of culture using an ELISA assay as shown in (B).

[0947] Figure 6: Anti-PD-1 / anti-CD28 with various configurations specifically activates PD1+ CD28+. (A) PD-1 binding ELISA assay. Human recombinant PD-1 protein was immobilized and different concentrations of anti-PD-1 / anti-CD28 multifunctional molecules configurations A1, B1, B2, C1 or C2 were added. Unveiling was performed using anti-human Fc antibodies coupled to peroxidase. Colorimetry was performed at 450 nm using TMB substrate. Data were normalized by anti-PD-1*2 or anti-PD-1*1 at 20 nM and considered as 100% binding for bivalent and monovalent plots, respectively. N=2 independent experiments per condition. (B) Antagonist PD-1 / PDL1 ELISA assay. Human recombinant PD-L1 protein was immobilized, and anti-PD-1 / anti-CD28 multifunctional molecular configurations A1, B1, B3, C1, C2, C5 were mixed with a fixed concentration of biotinylated human PD-1 at different concentrations and added to PD-L1 immobilized on an ELISA plate. Revealed with streptavidin coupled to peroxidase. Colorimetric assay was performed at 450 nm using TMB substrate. The formula for determining the percentage of antagonistic activity was 100-((DO450nM*100) / DO450nM without Ab). N=2 independent experiments. (C) CD28 binding MSD assay. Biotinylated human recombinant CD28 protein was immobilized on a streptavidin-coated plate, and different concentrations of anti-PD-1*1 / anti-CD28*1 multifunctional molecular configurations A1, B1, or B2 were added. Bivalent anti-CD28 binding domain and monovalent anti-PD-1 binding domain were loaded as positive and negative controls, respectively. Unveiling with anti-human Fc antibody coupled to SulfoTag. Electrical signals were measured using an MSD system. (D) Bioassay based on cis-active Jurkat T cells. IL-2 secretion bioassay of PD-1+CD28+ or PD-1-CD28+ Jurkat cells treated with anti-PD-1 / anti-CD28 multifunctional molecules. Briefly, PD-1+ CD28+ Jurkat cell lines were incubated on CD3-coated plates (2,5-3 µg / mL) in the presence of PD-1 / anti-CD28 multifunctional molecule configurations A1, B1, B2, B3, B4, C1, C2, C5 and C6 (100 nM), anti-PD-1 or anti-CD28 antibodies (bivalent anti-CD28 positive control for non-specific PD-1-independent activation). After 48 h of stimulation, IL-2 secretion in the culture supernatant was quantified and data were normalized by the condition without antibody. N=2-7 independent experiments. IL-2 secretion was normalized to the no antibody control condition. Statistical analysis was performed using the Mann-Whitney test: ** indicates p value ≤ 0.001, ns indicates not significant. (E) Bioassay based on cis-active Jurkat T cells pre-blocked with anti-CD28.Bioassay of IL-2 secretion by PD-1+CD28+ Jurkat cells after treatment with anti-CD28 antagonists and anti-PD1 / anti-CD28 multifunctional molecules. Briefly, PD-1+CD28+ Jurkat cell lines were treated with 1 µg / mL anti-CD28 antagonists for 20 minutes and then incubated on CD3-coated plates (3 µg / mL) in the presence of configuration B1 PD-1*1 / anti-CD28*1 scFv clone 1 (100 nM). After 48 hours of stimulation, IL-2 secretion in culture supernatants was quantified and data were normalized by conditions without antibody. N = 3 independent experiments and 2 additional experiments without anti-CD28 antagonists. Statistical analysis was performed using the Mann-Whitney test: * indicates p value ≤ 0.05. (F) Assay based on trans-active Jurkat T cells. PD-1 recombinant protein was coated and PD-1-CD28+ Jurkat T cells were incubated with 100 nM of PD-1 / anti-CD28 multifunctional molecule configurations A1, B1, B2, B4, C1, C2 and C6 (100 nM), anti-PD-1 or anti-CD28 antibodies (bivalent anti-CD28 positive control for nonspecific PD-1-independent activation). IL-2 secretion in the supernatant after 48 hours of culture was quantified using ELISA assay and data were normalized by the condition without antibody. N=1-2 independent experiments.

[0948] Figure 7: Other anti-CD28 clones in the prior art do not induce cis-activation of PD-1+CD28+ T cells, but Activation of CD28+PD1-negative T cells(A) Schematic diagram of configuration B1 anti-PD-1*1 / anti-CD28*1 scFv antibodies (B) Bioassay based on cis-active Jurkat T cells. Bioassay of IL-2 secretion from PD-1+ CD28+ or PD-1-CD28+ Jurkat cells treated with anti-PD-1 / anti-CD28 multifunctional molecules. Briefly, PD-1+ CD28+ Jurkat cell lines were incubated on CD3-coated plates (2,5-3 µg / mL) in the presence of configuration B1 anti-PD-1*1 / anti-CD28*1 scFv clone 1, clone 2 or clone 3 (100 nM). After 48 h of stimulation, IL-2 secretion in the culture supernatant was quantified and the data were normalized by the condition without antibody. N=2-6 independent experiments. (C) Assay based on trans-active Jurkat T cells. PD-1-CD28+ Jurkat T cells were coated with anti-CD3 and PD-1 recombinant proteins and incubated with 100 nM of configuration B1 anti-PD-1*1 / anti-CD28*1 scFv clone 1, clone 2, or clone 3 (100 nM). After 48 hours of culture, IL-2 secretion in the supernatant was quantified using ELISA and the data were normalized by the condition without antibody. N=1-2 independent experiments. (D) Bioassay based on cis-active Jurkat T cells. IL-2 secretion bioassay of PD-1+CD28+ or PD-1-CD28+ Jurkat cells treated with anti-PD1 / anti-CD28 unmutated or mutated clone 1. Briefly, PD-1+ CD28+ Jurkat cell lines were incubated on CD3-coated plates (2,5-3 µg / mL) in the presence of configuration B1 anti-PD-1*1 / anti-CD28*1 scFv clone 1 or mutant sequences mutVH_FR1, mutVH_FR2, mutVH_FR3, mutVL_FR1, mutVL_F2, mutVH_CDR1, mutVL_CDR1, mutVL_CDR2 (300 nM). After 48 hours of stimulation, IL-2 secretion in the supernatant was quantified and data were normalized by the condition without antibody and the fold change of normalized IL-2 secretion of PD1+ and PD-1- cells was done. N=2 independent experiments.

[0949] Figure 8 :Anti-CD28 / anti-PD-1 constructed with other anti-PD-1 clones and anti-CD28 clone 1 can cis-activate PD-1+ CD28+ T cells.Bioassay based on cis-active Jurkat T cells. IL-2 secretion bioassay of PD-1+ CD28+ or PD-1- CD28+ Jurkat cells treated with anti-PD-1 / anti-CD28 multifunctional molecules. Briefly, PD-1+ CD28+ Jurkat cell lines were incubated on CD3-coated plates (3 µg / mL) in the presence of anti-PD-1*1 / anti-CD28*1 scFv multifunctional molecule configuration B1 (100 nM) with monovalent anti-PD-1 clone 1 sequence or monovalent pembrolizumab sequence, monovalent anti-PD-1 (clone 1 sequence) or anti-CD28 antibodies (bivalent anti-CD28 positive control for nonspecific PD-1-independent activation). After 48 h of stimulation, IL-2 secretion in the culture supernatant was quantified and data were normalized by the condition without antibody. N=4 independent experiments.

[0950] Fig. 9 : Configuration B1 anti-PD-1*1 / anti-CD28*1 scFv reactivates human tumor-infiltrating lymphocytes. IFNy secretion by TILS. Freshly isolated lung tumors (n=2) and ovarian ascites (n=1) were processed to isolate and culture tumoroids. Tumoroids were cultured in triplicate at 37°C 5% C02 for 48 h in the presence of 70 nM of isotype control, PD-1*2, or anti-PD-1*1 / anti-CD28*1 scFv (Multifunctional Molecular Configuration B1 Clone 1). After incubation, supernatants were collected and IFNy secretion by TILS was quantified by MSD or ELISA. Statistical analysis was performed using the Wilcoxon test: * indicates p value ≤ 0.05.

[0951] Fig.10 : Anti-PD-1 / anti-CD28 multifunctional molecules with various configurations have good pharmacokinetics in mice. C57BL / 6Jrj mice received a single intravenous dose of 4 mg / kg or 5 mg / kg anti-PD-1 / anti-CD28 clone 1 multifunctional molecule (configuration A1, B1, B3, C2 and C6) or bivalent anti-PD-1. Blood samples were collected at 0.5, 1, 4, 24, 48, 100 and 144 hours. Antibody concentrations were determined in serum by ELISA using mouse anti-human κ IgG antibody and revealed with donkey anti-human IgG-peroxidase and TMB substrate. Example

[0952] Further aspects and advantages of the present invention will be described in the following examples which are to be regarded as illustrative rather than limiting.

[0953] Materials and methods

[0954] Multifunctional molecules

[0955] Figure 1 and Fig. 7A Examples of structures of multifunctional molecules according to the present invention are provided.

[0956] Anti-PD-1 Clone 1 is derived from the OSE-279 antibody disclosed in patent application WO2020 / 127366. This anti-PD-1 binding domain comprises a variable heavy chain as described in SEQ ID NO: 15 and a variable light chain as described in SEQ ID NO: 16.

[0957] Pembrolizumab is disclosed in patent application WO2008156712. The anti-PD-1 binding domain derived from pembrolizumab comprises a variable heavy chain as described in SEQ ID NO: 79 and a variable light chain as described in SEQ ID NO: 80.

[0958] Anti-CD28 Clone 1 specifically comprises the variable heavy chain set forth in SEQ ID NO: 44 and the variable light chain set forth in SEQ ID NO: 45.

[0959] Anti-CD28 Clone 2 is derived from patent application WO2006050949. Multifunctional molecules comprising Anti-CD28 Clone 2 are described in particular i) SEQ ID NO: 81 (anti-CD28 arm) and ii) SEQ ID NO: 25 and SEQ ID NO: 16 (anti-PD-1 arm). Such multifunctional molecules adopt the B1 configuration.

[0960] Anti-CD28 Clone 3 is derived from patent application WO2021260064. Multifunctional molecules comprising Anti-CD28 Clone 3 are described in particular in i) SEQ ID NO: 82 (anti-CD28 arm) and ii) SEQ ID NO: 25 and SEQ ID NO: 16 (anti-PD-1 arm). Such multifunctional molecules adopt the B1 configuration.

[0961] Anti-CD28 clone 1 mutant

[0962] Mutants of anti-CD28 clone 1 have been generated. In the mutant sequences provided in Table 2 below, the mutated amino acids are indicated in bold underline compared to the unmutated sequence. For each mutant, if the VH sequence comprises a mutation (e.g., as described in SEQ ID NO: 83, 84, 85, or 88), the VL sequence is unmutated (i.e., as described in SEQ ID NO: 45). Conversely, if the VL sequence comprises a mutation (e.g., as described in SEQ ID NO: 86, 87, 89, or 90), the VH sequence is unmutated (i.e., as described in SEQ ID NO: 44).

[0963]

[0964]

[0965] Table 2. hCD28 binding of anti-CD28 / anti-PD-1 multifunctional molecules (configuration B1) and anti-CD28 bivalent molecules determined using Biacore technology. Different constructs of configuration B1 anti-PD-1*1 / anti-CD28*1 scFV were generated using anti-CD28 scFv clone 1 (unmutated), 3 different framework regions in the VH domain (mutVH_FRs1, mutVH_FRs2, mutVH_FRs3), 2 different framework regions in the VL domain (mutVL_FRs1, mutVL_FRs2), 1 CDR mutation in the VH domain (mutVH_CDRs1) or 2 CDR mutations in the VL domain (mutVL_CDRs1, mutVL_CDRs2).

[0966] In particular, when the mutation is located in the framework region of the anti-CD28 binding domain, when compared to the original sequence of the anti-CD28 clone 1, mutVH_FRs1 comprises amino acid substitutions in the heavy chain framework regions HFR1, HFR2 and HFR3; mutVH_FRs2 comprises amino acid substitutions in HFR1, HFR2, HFR3 and HFR4; mutVH_FRs3 comprises amino acid substitutions in HFR1, HFR3 and HFR4; mutVL_FRs1 comprises amino acid substitutions in the light chain framework regions LFR1, LFR2, LFR3 and LFR4; mutVL_FRs2 comprises amino acid substitutions in LFR1, LFR3 and LFR4.

[0967] When the mutation is located in the CDR of the anti-CD28 binding domain, when compared with the original sequence of anti-CD28 clone 1, mutVH_CDRs1 comprises an amino acid substitution in the heavy chain CDR1 (the sequence of HCDR1 is determined according to the IGMT method); mutVL_CDRs1 comprises an amino acid substitution in the light chain CDR1 (the sequence of LCDR1 is determined according to the IGMT or Kabat method); mutVL_CDRs2 comprises an amino acid substitution in the light chain CDR2 (the sequence of LCDR1 is determined according to the IGMT or Kabat method).

[0968] PD-1 ELISA binding

[0969] Recombinant hPD1 (Sino Biologicals, Beijing, China; No. 10377-H08H) was immobilized on a 96-well plate. The plate was in carbonate buffer (pH 9.2) at 0.5 μg / ml, and purified antibodies were added to measure binding. After incubation and washing, peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch; USA; No. 709-035-149) was added and revealed by conventional methods.

[0970] Binding analysis with PD1+ expressing CHO cells

[0971] CHO cells were transduced with human PD1 gene and selected for expression of PD1 receptor. 100 nM purified antibody was added to CHO PD1+. After incubation and washing, phycoerythrin-labeled anti-human IgG (Biolegend; No. 409303) was added and revealed by cell counting by conventional methods.

[0972] PD-1 / PD-L1 antagonist ELISA assay

[0973] Competitive ELISA assay by PD-1: PD-L1 inhibitor screening ELISA assay pair (AcroBiosystems; USA; No. EP-101). In this assay, recombinant human PDL1 was immobilized on plastic at 2 μg / ml in PBS pH7.4 buffer. Purified antibodies (different concentrations) were mixed with biotinylated human PD1 (AcroBiosystems; USA; No. EP-101) at a final concentration of 0.66 μg / ml to measure competitive binding at 37°C for 2 h. After incubation and washing, peroxidase-labeled streptavidin (Vector laboratoring; USA; No. SA-5004) was added to detect biotin-PD-1Fc binding and revealed by conventional methods.

[0974] Measuring CD28 binding using MSD technology

[0975] For the activity ELISA assay, recombinant human CD28 (bio-techne; No. 342-CD-200) was immobilized on plastic at 5 μg / ml in PBS 1X (pH 7.4), and purified antibodies and multifunctional molecules were added to measure binding. After incubation and washing, sulfoTag-labeled monoclonal mouse anti-human kappa antibody (clone NaM76-5F3, labeled with MSD GOLD SULFO-TAGNHS-Ester reagent (MSD; No. # R91AO-1)) was added and revealed by the MSD system.

[0976] Jurkat phenotypic analysis

[0977] Jurkat T cell lymphoma was phenotyped using flow cytometry. Jurkat was incubated on ice with 1xyellow live dead (#L34968 Thermofisher) for 20 minutes. The cells were washed three times with PBS1X 2mM EDTA 2%FBS. Jurkat was incubated with anti-CD28-FITC (1 / 20; #555728 BD) on ice for 30 minutes. The cells were washed three times with PBS1X2mM EDTA 2%FBS. Jurkat was analyzed on a classic flow cytometer Cytoflex (Beckman). CD28 expression was studied with live (LIVE DEAD negative) single events using unstained and / or FMO (fluorescent minus one) controls.

[0978] Cis-activity bioassay

[0979] Jurkat T cell lymphoma expressing CD28 was transduced with human PD-1, resulting in stable expression of human PD-1 on the cell surface. For bioassay, Jurkat CD28+ PD-1- or Jurkat CD28+ PD-1+ were cultured on CD3-coated plates (2,5-3 ug / mL OKT3) in the presence of anti-PD-1 / anti-CD28 multifunctional molecules. After 48 hours of incubation, IL-2 secreted by Jurkat cells in the supernatant was quantified using ELISA (Duoset R&D kit #DY202-05 or OptEIA™ BD kit #555190).

[0980] Trans-activity bioassay

[0981] Jurkat CD28+ PD-1- cells were cultured on plates coated with CD3 (2,5-3 ug / mL OKT3) + human PD-1 in the presence of anti-PD-1 / anti-CD28 multifunctional molecules. After 48 hours of incubation, IL-2 secreted by Jurkat cells in the supernatant was quantified using ELISA (Duoset R&D kit #DY202-05 or OptEIA™ BD kit #555190).

[0982] Biacore

[0983] hCD28 recombinant protein (bio-techne, No. 342-CD-200) was immobilized on a CM5 sensor chip in sodium acetate at pH 5 in a BiacoreT200 at 20 ug / mL. Binding was measured by flowing increasing doses (0, 187, 375, 750, 1500 and 3000 nM) of anti-CD28 bivalent antibody or anti-CD28 / anti-PD-1 (configuration B1) antibody at a flow rate of 40 µL / min in HBS-EP buffer (provided by CYTIVA). Antigen-antibody binding kinetics were tracked for 2 minutes, and dissociation kinetics were tracked for 10 minutes. The binding and dissociation curves were fitted to the bivalent model.

[0984] Mouse Pharmacokinetics

[0985] Nine-week-old female C57BL6RJ wild-type mice were intravenously injected with a single dose of anti-PD-1 / anti-CD28 (4 mg / kg) or anti-PD-1*2 (5 mg / kg). After administration, blood was collected at multiple time points (0.5, 1, 4, 24, 48, 72, 100, and 144 hours), diluted 50-fold in PBS, centrifuged, and the antibody-containing supernatant (serum) was stored at -20°C (short-term) or -80°C (long-term) until quantitative analysis.

[0986] Drug concentrations in serum were determined by ELISA using immobilized mouse anti-human kappa IgG (clone NaM76-5F3) diluted to 1 μg / ml in 1× borate buffer. Detection was performed using peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch; USA; No. 709-035-149) and revealed by conventional methods.

[0987] Quantification of TILS-IFNy secretion in human tumors

[0988] After resection surgery, tumors were freshly collected and stored in MACS tissue storage containers (Miltenyi) at 2-8°C until processing (maximum 24 h after surgery). Two lung cancer samples were collected for this study. Ovarian ascites were freshly collected after puncture; one ovarian ascites was collected for this study. Samples were collected from patients treated with chemotherapy or radiotherapy, none of whom were treated with immune checkpoint blockade therapy.

[0989] Tumors were minced into fragments of 2-3 mm in 50% RPMI1640 (Gibco) 50% TexMacs (Miltenyi) supplemented with 5% FBS 100 U / mL penicillin and 0.1 mg / mL streptomycin / Primocin. 3. The fragments were plated evenly in ultra-low attachment plates. 70 nM antibodies (isotype control, anti-PD-1*2, and anti-PD-1*1 / anti-CD28*1 scFv configuration B1 clone 1) were added to the tumoroids (in triplicate) and the plates were incubated at 37°C, 5% CO2. After 48 h of culture, the culture supernatants were collected and stored at -80°C until MSD IFNy quantification.

[0990] After centrifugation of the ascites, the pellet was washed with 5 mM EDTA. After a second centrifugation, red blood cells were removed with RBC lysis buffer. The remaining cells were suspended in 50% RPMI 1640 (Gibco) 50% TexMacs (Miltenyi) supplemented with 5% FBS 100 U / mL penicillin and 0.1 mg / mL streptomycin / Primocin and plated in ultra-low attachment plates. 70 nM antibodies (isotype control, anti-PD-1*2, and anti-PD-1*1 / anti-CD28*1 scFv configuration B1 clone 1) were added to the cells (in triplicate) and the plates were incubated at 37°C, 5% CO2. After 48 h, the culture supernatant was collected and stored at -80°C until quantification by MSD. IFNy secreted by TILS in tumoroids was quantified by the S-plex human IFN-y kit (#K151X9S-2 MSD).

[0991] result

[0992] Example 1. Anti-PD-1 / anti-CD28 multifunctional molecules effectively constructed with monovalent anti-PD-1 effectively bind and antagonize Human PD-1 inhibitory signaling.

[0993] Binding of anti-PD-1 / anti-CD28 (configuration B1) to human PD-1 receptor was evaluated by ELISA. Briefly, recombinant human PD-1 receptor was coated on plates, multifunctional molecules were added in serial dilutions, and then revealed with secondary anti-human IgG antibody coupled to peroxidase + TMB. Binding to cells expressing PD-1 was also evaluated after incubation of human PD-1 transduced CHO cells with anti-PD-1 / anti-CD28 + anti-human IgG-PE antibodies. Antagonistic activity of multifunctional molecules against PD-1 was evaluated by competitive ELISA assay (Acrobiosystem).

[0994] like Figure 2 As shown, configuration B1 constructed with monovalent anti-PD-1 binds to the human PD-1 receptor and effectively antagonizes the interaction of PD-L1, indicating that even the anti-PD-1 domain of the multifunctional molecule can target PD-1+ exhausted T cells even in a monovalent configuration and block the engagement of PD-L1 / PD-1, thereby antagonizing PD-1-mediated inhibitory signaling and promoting the recovery of PD-1+ exhausted T cells (enhancing effector function).

[0995] Example 2. Anti-PD-1 / anti-CD28 multifunctional molecules effectively constructed with monovalent anti-CD28 can bind to human CD28

[0996] Figure 3 Anti-PD-1 / anti-CD28 (configuration B1) binding to human CD28 as assessed by ELISA using MSD technology is shown. Briefly, biotinylated human recombinant CD28 protein was immobilized on a streptavidin plate for MSD and different concentrations of anti-PD-1*1 / anti-CD28*1 scFv configuration B1 were added. Revealed with anti-human Fc antibody coupled to SulfoTag. The electrical signal was quantified using the MSD system.

[0997] Example 3. Anti-PD-1 / anti-CD28 scFv multifunctional molecule (configuration B1) cis-targets and specifically activates PD-1 + CD28+ T cells, while the remaining PD-1 negative CD28+ T cells

[0998] The cis efficacy of anti-PD-1 / anti-CD28 multifunctional molecules was evaluated in a cell-based bioassay using Jurkat T cells expressing either CD28 receptor alone or co-expressing CD28 and PD-1 receptors. Following treatment with anti-PD-1 / anti-CD28 (configuration B1) (2.5 ug / mL) on anti-CD3 coated plates, IL-2 secretion in the supernatant was quantified after 48 hours of incubation using an ELISA assay.

[0999] Figure 4 A, B, and C show anti-PD-1 / anti-CD28 (configuration B1). The multifunctional molecule promotes T cell activation (IL-2 secretion) in PD-1+ CD28+ T cells, while the drug has no effect on PD-1-negative T cells, demonstrating that the cis-specific activity of the molecule (i.e., for the same cells) triggers CD28 signaling only in PD-1+ T cells. As shown in Figure C, this effect is dose-dependent. At the same time, the inventors also showed that anti-PD-1 bivalent antibodies do not induce T cell activation, while bivalent anti-CD28 promotes the activation of all T cell populations, regardless of PD-1 expression. Since CD28 is expressed on all T cells, bivalent anti-CD28 antibodies will trigger the activation of all T cells, thereby inducing high immunotoxicity and cytokine storms, as described in previous clinical trials (Suntharalingam, et al., NEJM 2016). In contrast, the anti-PD-1 / anti-CD28 multifunctional molecules described by the inventors exhibit selective activation of PD-1+ T cells (tumor-specific exhausted T cells) without activating PD-1-negative T cells (naive CD28+ nonspecific T cells), which would prevent the nonspecific toxicity induced by bivalent anti-CD28 antibodies.

[1000] To confirm that the effects induced by the anti-PD1 / anti-CD28 multifunctional molecule are mediated only in cis mode and that the drug has no trans activity, the inventors investigated the effects of the drug on PD-1 coated plates to bind the bispecific anti-PD-1 domains, e.g. Figure 5 As shown in A. Jurkat T cells expressing only the CD28 receptor were then added to the plate, and the secretion of IL-2 in the supernatant was measured by ELISA after 48 h of co-incubation. Figure 5 B demonstrates that when PD-1 is coated on the plate, the anti-PD-1 / anti-CD28 (configuration B1) multifunctional molecule does not induce IL-2 secretion, indicating that the multifunctional molecule has no trans-active effect on PD-1 negative CD28+ T cells. These data confirm that the anti-PD-1 / anti-CD28 described by the inventors does not activate PD-1 negative cells (naive T cells) and only has the cis-dependent property of entering the same cells expressing the PD-1 receptor by triggering the CD28 signal.

[1001] In summary, the anti-PD-1 / anti-CD28 multifunctional molecules described by the inventors will act in a cis-dependent manner, by triggering CD28 co-stimulatory signals, while also restoring PD-1+CD28+ exhausted T cell effector functions by blocking PD-1 inhibitory signaling, without activating non-specific T cells. In contrast, bivalent anti-CD28 antibodies or other anti-CD28 bispecific antibodies described by others, with monovalent CD28 and monovalent anti-TAA domains, will transactivate PD-1 tumor-specific T cells as well as PD-1 negative non-specific T cells among all T cells.

[1002] Example 4. Anti-PD-1 / anti-CD28 multifunctional molecules of various configurations effectively and specifically activate in cis PD1+ CD28+ cells, while the remaining PD1- CD28+ cells

[1003] In addition to those described in Example 2, other configurations of anti-PD-1 / anti-CD28 multifunctional molecules were constructed using bivalent anti-PD-1 antibodies fused to anti-CD28 scFv (configurations C1 and C2), monovalent anti-PD-1 antibodies fused to anti-CD28 scFV (configurations B1 and B2), or monovalent anti-PD-1 Fab fused to monovalent anti-CD28 Fab CrossMAb (configuration A1), as described in Example 2. Figure 1 As described. These different constructs were generated with anti-CD28 clone 1. The ability of the antibodies to cis-activate PD-1+CD28+ T cells was assessed using the PD1+CD28+ Jurkat T cell bioassay. As detailed in Examples 2 and 3, the ability of the antibodies to bind to human CD28 (MSD technology) or human PD-1 (ELISA assay) was studied. After coating human PD-1 recombinant protein on an ELISA plate, the binding of anti-PD-1 / anti-CD28 multifunctional molecules to human PD-1 was assessed by ELISA.

[1004] Fig. 6A It was shown that bispecific antibodies with different configurations of bivalent (configurations C1 and C2) or monovalent (configurations B1 and B2) anti-PD-1 domains bind effectively to the human PD1 receptor. Antibodies using CrossMAb technology against the anti-CD28 domain (configuration A1) can bind to PD-1, but with lower potency. Fusion of the scFv anti-CD28 domain to the C-terminal or N-terminal domain of the anti-PD-1 binding domain did not alter the ability to bind to the PD-1 receptor, as similar binding activity was observed between configurations C1 and C2. The antagonistic activity of the anti-PD-1 / anti-CD28 multifunctional molecule against the PD-L1 / PD-1 axis was studied using a competitive ELISA assay (Accrobiosystems) and compared with two other configurations of scFv anti-CD28 fused to the C-terminus of a bivalent anti-PD1 (configuration C5) or a monovalent anti-PD1 (configuration B3).

[1005] Figure 6B The results showed that all configurations had potent antagonist anti-PD-1 activity. Configuration C5 showed higher antagonist activity, and the anti-PD-1 monovalent configurations (B1 and B3) showed similar antagonist activity to the bivalent anti-PD-1 constructs (C1 and C2).

[1006] Next, the inventors evaluated the binding properties of the different constructs to human CD28. The anti-PD-1*1 construct was used as a negative control sample for this assay.

[1007] Figure 6C All bispecific molecules with different configurations, either containing anti-CD28 scFv configurations (configurations B1 and B2) or with anti-CD28 Fab configurations (configuration A1), were validated to bind to human CD28. The orientation of the VH and VL domains did not affect binding to human CD28, as the B1 and B2 constructs had equivalent binding capacity. All anti-PD-1 / anti-CD28 constructs containing monovalent anti-CD28 targeting domains had lower binding potency to human CD28 than the anti-CD28 bivalent clone 1 antibody without anti-PD-1 valency (positive control of the experiment) ( Figure 6CAnd Table 1 below). For the bivalent anti-CD28 antibody, an affinity of 75.4 nM was observed, and for the anti-PD-1 / anti-CD28 antibody (configuration B1), the estimated KD was up to 5000 nM. Bivalent anti-CD28 antibodies can induce nonspecific T cell activation (independent of TCR triggering) and high toxicity in human patients, leading to nonspecific activation of naive T cells and high cytokine release. In order to specifically activate PD-1+ T cells and avoid triggering / aggregation of CD28 receptors on naive T cells that are independent of PD-1, the inventors designed an anti-PD-1 / anti-CD28 multifunctional molecule with a monovalent anti-CD28 configuration with low CD28 binding properties, so as to facilitate the multifunctional molecule to target the PD-1 receptor and strictly reactivate CD28 signaling in PD-1+ tumor-specific T cells.

[1008]

[1009] Table 1. Binding of hCD28 to anti-CD28 / anti-PD-1 multifunctional molecule (configuration B1) and anti-CD28 bivalent determined using Biacore technology. hCD28 recombinant protein (bio-techne, No. 342-CD-200) was immobilized on a CM5 sensor chip, and then anti-CD28 bivalent antibody or anti-CD28 / anti-PD-1 (configuration B1) multifunctional molecule was added in increasing doses, and kd and ka were measured.

[1010] The cis activity of the different constructs was assessed using the cell-based bioassay described in Example 3 by quantifying IL-2 secretion from PD-1+ CD28+ cells or PD-1 negative CD28+ Jurkat cells. Fig.6D The results show that all tes...

Claims

1. A multifunctional molecule comprising: (a) a first binding moiety that binds to a target specifically expressed on the surface of a T cell, the target being selected from the group consisting of PD-1, VISTA, CTLA-4, BTLA, TIGIT, CD160, LAG3, and TIM3; and (b) a second binding moiety which has an agonistic effect on CD28 and is an anti-CD28 antigen binding domain comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) and optionally a heavy chain constant domain (CH1) and a light chain constant domain (CL), wherein: (i) the heavy chain variable domain (VH) comprises complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, and (ii) the light chain variable domain (VL) comprises complementarity determining regions (CDRs) LCDR1, LCDR2 and LCDR3, in: - a heavy chain CDR1 (HCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 30, optionally with one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof; - a heavy chain CDR2 (HCDR2) comprising or consisting of the amino acid sequence of SEQ ID NO: 31, optionally with one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof; - a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 32; optionally having one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof; - the light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 33, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; - the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 34, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; and - the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 35, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; The multifunctional molecule comprises a single anti-CD28 antigen binding domain.

2. The multifunctional molecule of claim 1, wherein the target specifically expressed on the surface of T cells is PD-1.

3. The multifunctional molecule of claim 1 or 2, wherein the first binding moiety is a first antigen binding domain, the first antigen binding domain comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) and optionally a light chain constant domain (CL) and a first heavy chain constant domain (CH1), and wherein the first antigen binding domain is linked to the N-terminus of a first Fc chain, and the molecule further comprises a second Fc chain forming an Fc domain with the first Fc chain.

4. The multifunctional molecule of any one of claims 1 to 3, wherein the anti-CD28 antigen binding domain is Fab, CrossMAb or scFv, preferably scFv.

5. The multifunctional molecule of any one of claims 1 to 4, wherein the anti-CD28 antigen binding domain is a scFv and comprises: - From N-terminus to C-terminus, VH is linked to VL, preferably via a peptide linker; or - From N-terminus to C-terminus, VL is linked to VH, preferably via a peptide linker.

6. The multifunctional molecule of any one of claims 1 to 5, wherein: (i) the heavy chain variable domain (VH) further comprises heavy chain variable region framework regions (HFRs) HFR1, HFR2, HFR3 and HFR4, which respectively comprise or consist of the amino acid sequences of SEQ ID NOs: 36, 37, 38 and 39, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, and (ii) the light chain variable domain (VL) further comprises framework regions (LFR) LFR1, LFR2, LFR3 and LFR4, which respectively comprise or consist of the amino acid sequences of SEQ ID NOs: 40, 41, 42 and 43, optionally with one, two or three modifications selected from substitution, addition, deletion and any combination thereof.

7. The multifunctional molecule of any one of claims 1 to 5, wherein the anti-CD28 antigen binding domain comprises: (a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 44, 83, 84, 85 and 88, optionally with one, two or three amino acid modifications selected from substitutions, additions, deletions and any combination thereof, preferably substitutions; and (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 86, 87, 89 and 90, optionally It has one, two or three amino acid modifications selected from substitution, addition, deletion and any combination thereof, preferably substitution.

8. The multifunctional molecule of any one of claims 1 to 7, wherein the anti-CD28 antigen binding domain comprises: (a) a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 44, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, preferably in the framework regions; and (b) a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 45, optionally There are one, two or three modifications selected from substitution, addition, deletion and any combination thereof, preferably in the framework regions.

9. The multifunctional molecule of any one of claims 1-8, wherein the anti-CD28 antigen binding domain is covalently linked to: (i) the C-terminus of CL of the first binding moiety, (ii) the C-terminus of CH1 of the first binding moiety, (iii) the N-terminus of VH of the first binding moiety, or (iii) the N-terminus of VL of the first binding moiety, optionally via a peptide linker.

10. The multifunctional molecule of any one of claims 1 to 9, wherein the molecule comprises a first Fc chain and a second Fc chain that together form an Fc domain, and the anti-CD28 antigen binding domain is covalently linked to the C-terminus of one of the Fc chains, preferably the first Fc chain that is covalently linked to the first antigen binding domain at its N-terminus, optionally via a peptide linker.

11. The multifunctional molecule of any one of claims 1-9, wherein the molecule comprises a first Fc chain and a second Fc chain that together form an Fc domain, the anti-CD28 antigen binding domain is covalently linked via its C-terminus to the N-terminus of the second Fc chain, and the first antigen binding domain is covalently linked via its C-terminus to the N-terminus of the first Fc chain, optionally via a peptide linker.

12. The multifunctional molecule of any one of claims 1 to 11, wherein the first binding moiety is or is derived from an anti-PD-1 antibody selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, cemiplizumab, carrelizumab, AUNP12, AMP-224, AGEN-2034, tislelizumab, PDR001, MK-3477, PF-06801591, JNJ-6372328 3. Genolizumab, LZM-009, BCD-100, SHR-1201, BAT-1306, AK-103, MEDI-0680, JS001, BI-754091, CBT-501, INCSHR1210, TSR-042, GLS-010, AM-0001, STI-1110, MGA012 or IBI308, preferably pembrolizumab or nivolumab.

13. The multifunctional molecule of any one of claims 1 to 11, wherein the first binding moiety is an anti-PD-1 antigen binding domain, which preferably comprises: (i) a VH comprising HCDR1, HCDR2 and HCDR3, and (ii) a VL comprising LCDR1, LCDR2 and LCDR3, in: - heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 1; - heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 2; - heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 3; - light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 4; - the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 5, and - the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO:

6.

14. The multifunctional molecule of any one of claims 1 to 11, wherein the first binding moiety is an anti-PD-1 antigen binding domain, preferably comprising: (a) VH comprising or consisting of the amino acid sequence of SEQ ID NO: 15, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof; preferably in the framework region; and (b) VL comprising or consisting of the amino acid sequence of SEQ ID NO: 16, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof, preferably in the framework region.

15. The multifunctional molecule of any one of claims 1-11, wherein the first binding moiety is an anti-PD-1 antigen binding domain comprising: a) (i) a VH comprising HCDR1, HCDR2 and HCDR3, and (ii) a VL comprising LCDR1, LCDR2 and LCDR3, wherein: the heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 65; the heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 66; the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 67; the light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 68; the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 69, and the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 70; or b) i) a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 71; and ii) a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 72; or c) (i) a VH comprising HCDR1, HCDR2 and HCDR3, and (ii) a VL comprising LCDR1, LCDR2 and LCDR3, wherein: the heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 73; the heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 74; the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 75; the light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 76; the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 77, and the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 78; or d) i) a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 79; and ii) a VL comprising or consisting of the amino acid sequence of SEQ ID NO:

80.

16. The multifunctional molecule of any one of claims 1-15, wherein the anti-PD-1 antigen binding domain is F(ab')2, Fab or CrossMAb.

17. An isolated nucleic acid sequence or a group of isolated nucleic acid sequences encoding a multifunctional molecule according to any one of claims 1 to 16.

18. A vector comprising the isolated nucleic acid sequence or the isolated nucleic acid sequence group according to claim 17.

19. A host cell comprising the vector of claim 18 or the isolated nucleic acid or nucleic acid molecule group of claim 17.

20. A method for producing a multifunctional molecule according to any one of claims 1 to 16, said method comprising the steps of culturing the host cell according to claim 19 and isolating said multifunctional molecule.

21. A pharmaceutical composition comprising the multifunctional molecule according to any one of claims 1 to 16, the nucleic acid or nucleic acid molecule group according to claim 17, the vector according to claim 18 or the host cell according to claim 19, and a pharmaceutically acceptable carrier.

22. The pharmaceutical composition of claim 21, the multifunctional molecule of any one of claims 1 to 16, the isolated nucleic acid or the set of nucleic acid molecules of claim 17, the vector of claim 18 or the host cell of claim 19 for use as a medicament.

23. The pharmaceutical composition, the multifunctional molecule, the isolated nucleic acid or the set of nucleic acid molecules, the vector or the host cell for use according to claim 22 for the treatment of cancer or the treatment of infectious diseases.

24. The pharmaceutical composition, multifunctional molecule, nucleic acid or nucleic acid molecule group, vector or host cell for use according to claim 23, wherein the disease is a cancer selected from the group consisting of brain cancer, carcinoma, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, lymphoma, glioma, mesothelioma, melanoma, gastric cancer, urethral cancer, environmentally induced cancers and any combination of said cancers.

25. The pharmaceutical composition, multifunctional molecule, nucleic acid or nucleic acid molecule group, vector or host cell for use according to claim 23, wherein the disease is an infectious disease caused by a virus selected from the group consisting of human immunodeficiency virus, hepatitis virus, herpes virus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, human T-lymphotropic virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus.

26. Pharmaceutical composition, multifunctional molecule, nucleic acid or set of nucleic acid molecules, vector or host cell for use according to any one of claims 22 to 25, for use in combination with radiotherapy or with another therapeutic agent, in particular a chemotherapeutic agent or an anti-infective agent.

27. The pharmaceutical composition, multifunctional molecule, nucleic acid or nucleic acid molecule group, vector or host cell for use according to claim 26, wherein the additional therapeutic agent is selected from the group consisting of a chemotherapeutic agent, an anti-infective agent, an alkylating agent, an angiogenesis inhibitor, an antibody, an antimetabolite, an anti-mitotic agent, an anti-proliferative agent, an antiviral agent, an Aurora kinase inhibitor, an apoptosis promoter, a death receptor pathway activator, a Bcr-Abl kinase inhibitor, a bispecific T cell engager antibody drug conjugate, a Bruton's tyrosine kinase (BTK) inhibitor, a cyclin-dependent kinase inhibitor, a cell cycle inhibitor, a cyclooxygenase-2 inhibitor, a leukemia virus oncogene homolog receptor inhibitor, a growth factor inhibitor, a heat shock protein-90 inhibitor, a histone deacetylase inhibitor, Hormonal therapy, apoptosis protein inhibitors, antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNA, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, non-steroidal anti-inflammatory drugs, poly ADP-ribose polymerase inhibitors, platinum chemotherapy agents, polo-like kinase inhibitors, phosphoinositide-3 kinase inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoids, small inhibitory ribonucleic acids (siRNA), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, peptide vaccines, cancer vaccines, epitopes or new epitopes from tumor antigens, and combinations of one or more of these agents.

28. A method for treating cancer or an infectious disease in a subject in need thereof, said method comprising administering the multifunctional molecule according to any one of claims 1 to 16 or the pharmaceutical composition according to claim 21, preferably in an effective amount.

29. Use of the multifunctional molecule according to any one of claims 1 to 16 or the pharmaceutical composition according to claim 21 in the preparation of a medicament for treating cancer or infectious diseases.

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