TIGIT-targeted single-domain antibody

Abstract

The invention provides a monoclonal antibody specifically combined with TIGIT and a bispecific antibody constructed on the basis of the TIGIT monoclonal antibody. The invention also provides a nucleic acid molecule for coding the antibody, an expression vector for expressing the antibody, a host cell and a preparation method. The invention also provides methods of treatment using the antibodies of the invention.

Description

[0001] This application is a divisional application of a Chinese patent application with the application number 202280006947.6, the application date of December 30, 2022, and the invention title of "TIGIT single-domain antibody and bispecific antibody based thereon".

[0002] The present invention provides a single-domain antibody that specifically binds to TIGIT. It also provides a nucleic acid molecule encoding the antibody, an expression vector for expressing the antibody, a host cell, and a preparation method. The present invention also provides a treatment method using the antibody of the present invention. Background Art

[0003] TIGIT (T-cell immunoreceptor with Ig and ITIM domains) belongs to the members of the immunoglobulin superfamily and is also known as VSTM3, WUCAM, or VSIG9. It is composed of an extracellular immunoglobulin V-like domain (IgV domain), a type I transmembrane domain, a tyrosine-based immunoreceptor inhibitory motif (ITIM), and an immunoglobulin tyrosine tail (ITT)-like motif (Jinah Yeo, Minkyung Ko, et al. (2021). "TIGIT / CD226 Axis Regulates Anti-Tumor Immunity" Pharmaceuticals 14(3):200.). TIGIT is mainly expressed in effector CD4+ T cells, follicular helper CD4+ T cells, regulatory T cells (Tregs), effector CD8+ T cells, and NK cells, and has become a popular target for cancer immunotherapy.

[0004] Multiple ligands of TIGIT have been identified, including: PVR (Necl-5 or CD155), Nectin2 (CD112), Nectin3 (CD113), and Nectin4 (PVRL4). However, the interaction between TIGIT and CD155 is the strongest, with an reported affinity of approximately 1 nM. The affinities for Nectin2 and 3 are very low, and the affinity for Nectin4 is close to that for CD155 (Reches A., Ophir Y., et al. (2020). "Nectin4 is a novel TIGIT ligand which combines checkpoint inhibition and tumor specificity" J. Immunother. Cancer. 8.). These ligands are mainly overexpressed on APC cells and various malignant tumor cells (such as colorectal cancer, melanoma, etc.). CD155 plays an immunomodulatory role through interactions with TIGIT, CD226, and CD96. Since the affinity of CD155 for TIGIT is much higher than that for CD226 and CD96, TIGIT and CD155 will preferentially bind, activating the inhibitory signals mediated by two motifs present in the cytoplasmic tail of TIGIT: the tyrosine-based immunoreceptor inhibitory motif (ITIM) and the immunoglobulin tyrosine tail (ITT)-like motif. Ligands on the surface of tumor cells bind to TIGIT on the surface of NK cells and T cells, inhibiting NK cell cytotoxicity and T cell activity, thereby mediating the immune escape mechanism of tumor cells. Karsten Mahnke et al. showed that since this inhibitory signaling pathway exists outside the classical PD1 / PD-L1 co-inhibitory pathway, the blockade of both signaling pathways by bispecific antibodies leads to a significant enhancement of the effector function of melanoma-specific cytotoxic T cells. The PD1 / PD-L1 signaling pathway axis has been confirmed in melanoma, and a second inhibitory pathway characterized by the TIGIT / CD155 interaction also exists in melanoma (Karsten Mahnke and Alexander H. Enk (2015). "TIGIT-CD155 Interactions in Melanoma: A Novel Co-Inhibitory Pathway with Potential for Clinical Intervention" Journal of Investigative Dermatology 136(1):9-11.).

[0005] Given that the inhibitory signaling pathway of TIGIT exerts a powerful inhibitory effect in different immune cell subsets, and its ligand CD155 is widely expressed in various solid tumors, targeting TIGIT is a very promising therapeutic strategy.

[0006] Since TIGIT is highly expressed on the surface of both T cells and NK cells, while other immune checkpoints such as PD1 are only expressed on the surface of T cells, this determines that TIGIT has greater advantages as a therapeutic target. Therefore, there is a need in the art to develop bispecific antibody drugs based on TIGIT antibodies. Summary of the Invention

[0007] The present invention provides an anti-TIGIT single-domain antibody (sdAb, single domain antibody), and a bispecific antibody constructed by applying it. In some embodiments, the anti-TIGIT single-domain antibody of the present invention has a very high affinity for human TIGIT and can recognize human and cynomolgus monkey TIGIT.

[0008] In some embodiments, the anti-TIGIT single-domain antibody can effectively block the binding of TIGIT to the PVR protein, and the blocking activity is significantly superior to that of the control antibody Tiragolumab.

[0009] The TIGIT single-domain antibody of the present invention can also effectively activate T cells to release cytokines.

[0010] The anti-PD1 / TIGIT bispecific antibody of the present invention has blocking activity against both TIGIT / CD155 and PD1 / PD-L1, and has a bright therapeutic prospect in indications where both PD1 / PD-L1 and TIGIT / CD155 exist.

[0011] The anti-TIGIT / anti-CTLA4 bispecific antibody of the present invention has one or more of the following activities / functions: First, it can bind well to TIGIT and CTLA4, and has a higher affinity for TIGIT than for CTLA4 (for example, one order of magnitude higher), thereby causing the anti-TIGIT and anti-CTLA4 bifunctional antibody to localize to the tumor site, reducing the residence time of the bispecific antibody in the peripheral system, thus reducing the peripheral toxicity of the CTLA4 antibody end and increasing the dosage of the CTLA4 antibody; Second, TIGIT and CTLA4 are highly expressed on Treg cells in tumors, and the bifunctional antibody of the present invention can eliminate immunosuppressive Treg cells in tumors through the Fc effect; Third, the bifunctional antibody of the present invention can specifically relieve the immune suppression of TIGIT and CTLA4 on effector T cells, activate T cells, and thus play an anti-tumor role, having good application prospects.

[0012] In one aspect, the present invention relates to the following specific embodiments:

[0013] 1. A VHH antibody that specifically binds to TIGIT, comprising

[0014] the three complementarity determining regions (CDRs) contained in the VH shown in any one of SEQ ID NO: 1, 6, 9, 14, 16, 18, and 21,

[0015] Preferably, the CDR sequences are defined according to IMGT.

[0016] 2. The VHH antibody of embodiment 1, comprising complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, wherein

[0017] (i) VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4 or 23 or 57, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 5; or

[0018] (ii) VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 8, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4 or 23 or 57, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 5; or

[0019] (iii) VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 11, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 12, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 13.

[0020] 3. The VHH antibody of embodiment 1, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region

[0021] (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in any one of SEQ ID NO: 1, 6, 9, 14, 16, 18, and 21; or

[0022] (ii) comprises or consists of the amino acid sequence shown in any one of SEQ ID NO: 1, 6, 9, 14, 16, 18, and 21; or

[0023] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence shown in any one of SEQ ID NO: 1, 6, 9, 14, 16, 18, and 21, preferably, the amino acid alterations do not occur in the CDR regions.

[0024] 4. A heavy-chain antibody that specifically binds to TIGIT, comprising the VHH antibody according to any one of embodiments 1-3.

[0025] 5. The heavy-chain antibody of embodiment 4, comprising the VHH antibody according to any one of embodiments 1-3 linked to an antibody constant region or Fc region, preferably, the antibody constant region or Fc region is from human IgG1, human IgG2, human IgG3, or human IgG4, optionally, the VHH antibody is linked to the Fc region through a hinge region or a part thereof, preferably, the amino acid sequence of the hinge region part is EPKSS (SEQ ID NO: 43).

[0026] 6. The heavy-chain antibody of embodiment 4, comprising the VHH antibody according to any one of embodiments 1-3 linked to an antibody Fc region, wherein the Fc region is the Fc region from human IgG1 or IgG4, preferably, the Fc region

[0027] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 40 or 42; or

[0028] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 40 or 42; or

[0029] (iii) comprising an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 40 or 42.

[0030] 7. The heavy-chain antibody of embodiment 5 or 6, wherein the Fc region comprises mutations that improve the effector function of the Fc region, such as mutations that enhance ADCC. Preferably, the mutations are the following combination of mutations: S239D, A330L, and I332E (EU numbering). Preferably, it comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 41 or consists thereof, and comprises the following combination of mutations: S239D, A330L, and I332E (EU numbering).

[0031] 8. The heavy-chain antibody of embodiment 4, which

[0032] (i) comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in any one of SEQ ID NOs: 2, 7, 10, 15, 17, 19, 20, or 22 or consists thereof; or

[0033] (ii) comprises the amino acid sequence shown in any one of SEQ ID NOs: 2, 7, 10, 15, 17, 19, 20, or 22 or consists thereof; or

[0034] (iii) comprises an amino acid sequence having 1 or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence shown in any one of SEQ ID NOs: 2, 7, 10, 15, 17, 19, 20, or 22. Preferably, the amino acid alterations do not occur in the CDR regions.

[0035] 9. The VHH antibody of any one of embodiments 1-3, or the heavy-chain antibody of any one of embodiments 4-8, wherein the antibody is a chimeric antibody or a humanized antibody.

[0036] 10. A bispecific antibody that comprises a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds to TIGIT and comprises the VHH antibody of any one of embodiments 1-3 and 9, or the heavy-chain antibody of any one of embodiments 4-9.

[0037] 11. The bispecific antibody of embodiment 10, wherein the second antigen-binding region specifically binds to PD-1, PD-L1 or PD-L2 or CTLA-4. Preferably, the second antigen-binding region specifically binds to PD-1 and comprises the PD1 antibody from WO2019219064A or an antigen-binding fragment thereof, such as a single-chain Fv, Fab, Fab', (Fab)2, single-domain antibody, VHH or heavy-chain antibody of the anti-PD-1 antibody. Preferably, the second antigen-binding region specifically binds to CTLA-4 and comprises the Ipilimumab antibody or an antigen-binding fragment thereof, such as a single-chain Fv, Fab, Fab', (Fab)2, single-domain antibody, VHH or heavy-chain antibody of the anti-CTLA-4 antibody.

[0038] 12. The bispecific antibody of embodiment 10 or 11, wherein the VHH antibody is linked to the C-terminus of the Fc fragment of the second antigen-binding region, or linked to the N-terminus of the VH fragment of the heavy chain of the second antigen-binding region, or can be inserted between the Fab fragment (Fab fragment heavy chain) and the Fc fragment of the second antigen-binding region, i.e., linked to the C-terminus of the Fab fragment heavy chain and the N-terminus of the Fc fragment. Optionally, the first and second antigen-binding regions are linked by a linker, preferably the linker comprises the (GGS)n amino acid sequence, where n is an integer of 1, 2, 3, 4, or 5, preferably n = 1.

[0039] 13. The bispecific antibody of embodiment 12, wherein the bispecific antibody has the following structure:

[0040] Heavy chain: from the N-terminus to the C-terminus, the heavy-chain variable region VH of the second antigen antibody - heavy-chain constant region CH1 - heavy-chain constant region Fc - anti-TIGIT VHH; or

[0041] from the N-terminus to the C-terminus, the heavy-chain variable region VH of the second antigen antibody - heavy-chain constant region CH1 - anti-TIGIT VHH - heavy-chain constant region Fc; or

[0042] from the N-terminus to the C-terminus, anti-TIGIT VHH - the heavy-chain variable region VH of the second antigen antibody - heavy-chain constant region CH1 - heavy-chain constant region Fc;

[0043] Light chain: from the N-terminus to the C-terminus, the light-chain variable region of the second antigen antibody - light-chain constant region CL;

[0044] Preferably, the second antigen is selected from PD-1 or CTLA-4.

[0045] 14. The bispecific antibody of embodiment 10 or 11, wherein the bispecific antibody has the following structure:

[0046] Heavy chain 1: From the N-terminus to the C-terminus, variable heavy chain region VH of the second antigen antibody - constant heavy chain region CH1 - constant heavy chain region Fc;

[0047] Heavy chain 2: One or more (e.g., two) tandem anti-TIGIT VHH - constant heavy chain region Fc

[0048] Light chain: From the N-terminus to the C-terminus, variable light chain region of the second antigen antibody - constant light chain region CL;

[0049] Preferably, the second antigen is selected from PD-1 or CTLA-4, such as CTLA-4.

[0050] 15. The bispecific antibody of embodiment 14, wherein the anti-TIGIT-VHH is linked to the N-terminus of the constant heavy chain region Fc through a linker peptide, for example, the linker peptide is the hinge region or a part thereof from human IgG1, 2, 3, or 4, including the native or mutated hinge region or a part thereof, such as from the human IgG1 hinge region, for example, the linker peptide is EPKSS (SEQ ID NO:43).

[0051] 16. The bispecific antibody of embodiment 14 or 15, wherein when heavy chain 2 contains multiple tandem anti-TIGIT single-domain antibody VHHs, the individual tandem VHHs can be linked through a linker. Preferably, the linker contains the (GGGGS)n amino acid sequence, where n is an integer of 1, 2, 3, 4, or 5, preferably n = 1.

[0052] 17. The bispecific antibody of any one of embodiments 10-16, wherein the constant heavy chain region CH1 of the second antigen antibody is from IgG, such as IgG1, IgG2, IgG3, or IgG4; preferably, the constant heavy chain region CH1 is from IgG1 or IgG4, more preferably, the constant heavy chain region CH1 contains or consists of the amino acid sequence as set forth in SEQ ID NO:28 or 31.

[0053] 18. The bispecific antibody of any one of embodiments 10-17, wherein the second constant heavy chain region Fc is defined as in any one of embodiments 5-7.

[0054] 19. The bispecific antibody of embodiment 14, wherein the Fc region of heavy chain 1 is different from the Fc region of heavy chain 2.

[0055] 20. The bispecific antibody of embodiment 19, wherein a knob mutation and a hole mutation are respectively introduced into the Fc region of heavy chain 1 and the Fc region of heavy chain 2.

[0056] 21. The bispecific antibody of embodiment 21, wherein

[0057] The first Fc region contains a knob mutation, which

[0058] (i) comprises an amino acid sequence SEQ ID NO:78 or an amino acid sequence having at least 90% identity thereto, such as 95%, 96%, 97%, 99% or higher identity, or consists thereof; or

[0059] (ii) comprises an amino acid sequence having at least 90% identity to SEQ ID NO:78, such as 95%, 96%, 97%, 99% or higher identity, and comprises S239D, A330L and I332E mutations and a knot mutation (such as S354C and T366W); and

[0060] The second Fc region comprises a buckle mutation, which

[0061] (i) comprises an amino acid sequence SEQ ID NO:77 or an amino acid sequence having at least 90% identity thereto, such as 95%, 96%, 97%, 99% or higher identity, or consists thereof; or

[0062] (ii) comprises an amino acid sequence having at least 90% identity to SEQ ID NO:77, such as 95%, 96%, 97%, 99% or higher identity, and comprises S239D, A330L and I332E mutations and a buckle mutation.

[0063] 22. The bispecific antibody of embodiment 13, which comprises

[0064] Heavy chain: from the N-terminus to the C-terminus, the heavy chain variable region VH of the second antigen antibody - the heavy chain constant region CH1 - the heavy chain constant region Fc - anti-TIGIT VHH; and

[0065] Light chain: from the N-terminus to the C-terminus, the light chain variable region of the second antigen antibody - the light chain constant region CL,

[0066] wherein the second antigen is PD-1;

[0067] wherein the heavy chain

[0068] (i) comprises the amino acid sequence of SEQ ID NO:30 or 33, or

[0069] (ii) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence described in SEQ ID NO:30, 32 or 33, or

[0070] (iii) consists of the amino acids described in SEQ ID NO:30 or 33;

[0071] and / or

[0072] Light chain

[0073] (i) comprising the amino acid sequence of SEQ ID NO:39, or

[0074] (ii) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:39, or

[0075] (iii) consisting of the amino acids of SEQ ID NO:39.

[0076] 23. The bispecific antibody of embodiment 13, comprising

[0077] Heavy chain:

[0078] From the N-terminus to the C-terminus, the heavy chain variable region VH of the second antigen antibody - heavy chain constant region CH1 - anti-TIGIT VHH - heavy chain constant region Fc

[0079] Light chain: from the N-terminus to the C-terminus, the light chain variable region - light chain constant region CL of the second antigen antibody,

[0080] wherein the second antigen is PD-1;

[0081] wherein the heavy chain

[0082] (i) comprising the amino acid sequence of SEQ ID NO:32, or

[0083] (ii) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:32, or

[0084] (iii) consisting of the amino acids of SEQ ID NO:32;

[0085] and / or

[0086] Light chain

[0087] (i) comprising the amino acid sequence of SEQ ID NO:39, or

[0088] (ii) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:39, or

[0089] (iii) consisting of the amino acids of SEQ ID NO:39.

[0090] 24. The bispecific antibody of embodiment 13, which comprises

[0091] Heavy chain: from the N-terminus to the C-terminus, the heavy chain variable region VH of the anti-TIGIT VHH-second antigen antibody - the heavy chain constant region CH1 - the heavy chain constant region Fc;

[0092] Light chain: from the N-terminus to the C-terminus, the light chain variable region of the second antigen antibody - the light chain constant region CL,

[0093] wherein the second antigen is CTLA-4,

[0094] wherein the heavy chain

[0095] (i) comprises the amino acid sequence of SEQ ID NO:69, or

[0096] (ii) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence described in SEQ ID NO:69, or

[0097] (iii) consists of the amino acids described in SEQ ID NO:69;

[0098] and / or

[0099] light chain

[0100] (i) comprises the amino acid sequence of SEQ ID NO:68, or

[0101] (ii) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence described in SEQ ID NO:68, or

[0102] (iii) consists of the amino acids described in SEQ ID NO:68.

[0103] 25. The bispecific antibody of embodiment 14, which comprises

[0104] Heavy chain 1: from the N-terminus to the C-terminus, the heavy chain variable region VH of the second antigen antibody - the heavy chain constant region CH1 - the heavy chain constant region Fc;

[0105] Heavy chain 2: one or more (e.g., two) tandem anti-TIGIT VHH - heavy chain constant region Fc

[0106] Light chain: from the N-terminus to the C-terminus, the light chain variable region of the second antigen antibody - the light chain constant region CL,

[0107] wherein heavy chain 1

[0108] (i) comprising the amino acid sequence of SEQ ID NO:71, or

[0109] (ii) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:71, or

[0110] (iii) consisting of the amino acids of SEQ ID NO:71;

[0111] and / or

[0112] Heavy chain 2

[0113] (i) comprising the amino acid sequence of SEQ ID NO:72 or 74, or

[0114] (ii) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:72 or 74, or

[0115] (iii) consisting of the amino acids of SEQ ID NO:72 or 74;

[0116] and / or

[0117] Light chain

[0118] (i) comprising the amino acid sequence of SEQ ID NO:68, or

[0119] (ii) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:68, or

[0120] (iii) consisting of the amino acids of SEQ ID NO:68.

[0121] 26. A nucleic acid molecule encoding the VHH antibody of any one of embodiments 1-3 and 9, or the heavy chain antibody of any one of embodiments 4-9, or the heavy chain and / or light chain of the bispecific antibody of any one of embodiments 10-25, or consisting of said nucleic acid sequence.

[0122] 27. An expression vector comprising the nucleic acid molecule of embodiment 26, preferably, said expression vector is pCDNA, such as pCDNA3.1.

[0123] 28. A host cell comprising the nucleic acid molecule according to embodiment 26 or the expression vector according to embodiment 27. Preferably, the host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as 293FT cells or CHO-S cells.

[0124] 29. A method for preparing a VHH antibody according to any one of embodiments 1-3 and 9, or a heavy-chain antibody according to any one of embodiments 4-9, or a bispecific antibody according to any one of embodiments 10-25, the method comprising culturing a host cell according to any one of the embodiments comprising a nucleic acid encoding a VHH antibody or a heavy-chain antibody, or nucleic acids encoding the respective chains of a bispecific antibody, or an expression vector comprising the nucleic acid, under conditions suitable for the expression of the VHH antibody or the heavy-chain antibody or the bispecific antibody, and optionally recovering the VHH antibody or the heavy-chain antibody or the bispecific antibody from the host cell (or the host cell culture medium).

[0125] 30. An immunoconjugate comprising a VHH antibody according to any one of embodiments 1-3 and 9, or a heavy-chain antibody according to any one of embodiments 4-9, or a bispecific antibody according to any one of embodiments 10-25.

[0126] 31. A pharmaceutical composition or a drug or a preparation comprising a VHH antibody according to any one of embodiments 1-3 and 9, or a heavy-chain antibody according to any one of embodiments 4-9, or a bispecific antibody according to any one of embodiments 10-25, or the immunoconjugate according to embodiment 30 and optionally a pharmaceutically acceptable excipient.

[0127] 32. A pharmaceutical combination product comprising a VHH antibody according to any one of embodiments 1-3 and 9, or a heavy-chain antibody according to any one of embodiments 4-9, or a bispecific antibody according to any one of embodiments 10-25, or the immunoconjugate according to embodiment 30, and another therapeutic agent.

[0128] 33. A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of a VHH antibody according to any one of embodiments 1-3 and 9, or a heavy-chain antibody according to any one of embodiments 4-9, or a bispecific antibody according to any one of embodiments 10-25, or the immunoconjugate according to embodiment 30, or the pharmaceutical composition or preparation according to embodiment 31; or the pharmaceutical combination product according to embodiment 32.

[0129] 34. The method according to embodiment 33, wherein the cancer is a cancer characterized by having an elevated protein level and / or nucleic acid level (such as elevated expression) of PD-1, PD-L1 or PD-L2 and / or having an elevated protein level and / or nucleic acid level (such as elevated expression) of TIGIT.

[0130] 35. The method of embodiment 34, wherein the method further comprises co-administering in combination with other therapies such as treatment modalities and / or other therapeutic agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0131] Figure 1 Showing that the heavy chain antibody against human TIGIT blocks the binding of human TIGIT protein to CD155 (EC50, nM).

[0132] Figure 2 Showing the binding activity of the heavy chain antibody against human TIGIT to HEK293-human TIGIT cells (EC50, nM).

[0133] Figure 3 Showing the binding activity of the heavy chain antibody against human TIGIT to HEK293-cynomolgus monkey TIGIT cells (EC50, nM).

[0134] Figure 4 Showing that the heavy chain antibody against human TIGIT activates CD8+ T cells to release IFNγ factor.

[0135] Figure 5 Showing that the heavy chain antibody against human TIGIT after sequence optimization and Fc modification blocks the binding of human TIGIT protein to CD155 (IC50, nM).

[0136] Figure 6 shows the schematic structure of the anti-PD1 / TIGIT bispecific antibody of the present invention, Figure 6A which is a schematic diagram of E4, Figure 6B and is a schematic diagram of D1 and D4.

[0137] Figure 7 Showing the binding activity of the anti-PD1 / TIGIT bispecific antibody to human TIGIT protein (EC50, nM).

[0138] Figure 8 Showing the binding activity of the anti-PD1 / TIGIT bispecific antibody to human PD1 protein (EC50, nM).

[0139] Figure 9 Showing the activity of the anti-PD1 / TIGIT bispecific antibody in blocking the binding of human TIGIT protein to CD155 (IC50, nM).

[0140] Figure 10 Showing the activity of the anti-PD1 / TIGIT bispecific antibody in blocking the binding of human PD1 protein to PD-L1 (IC50, nM).

[0141] Figure 11 Showing the binding activity of the anti-PD1 / TIGIT bispecific antibody to HEK293-human TIGIT cells (EC50, nM).

[0142] Figure 12 Show the binding activity (EC50, nM) of the anti-PD1 / TIGIT bispecific antibody to Jurkat-NFAT-human PD1 cells.

[0143] Figure 13 Show the ADCC killing activity (EC50, nM) of the anti-PD1 / TIGIT bispecific antibody against activated CD4+ T or CD8+ T cells.

[0144] Figure 14 shows the schematic structure of the anti-TIGIT / CTLA-4 bispecific antibody of the present invention. Figure 14A It is a schematic diagram of THC4. Figure 14B It is a schematic diagram of CT1KH. Figure 14C It is a schematic diagram of CT2KH.

[0145] Figure 15 Show the activity of the anti-TIGIT / CTLA-4 bispecific antibody in blocking the binding of human TIGIT protein to CD155.

[0146] Figure 16 Show the activity of the anti-TIGIT / CTLA-4 bispecific antibody in blocking the binding of human CTLA-4 protein to CD80.

[0147] Figure 17 Show the binding activity of the anti-TIGIT / CTLA-4 bispecific antibody to HEK293-human TIGIT cells Invention Detailed description

[0148] I. Single-domain antibodies (VHH antibodies) that bind to TIGIT, heavy-chain antibodies, and bispecific antibodies containing the same

[0149] In a first aspect, the present invention relates to an antibody that binds to TIGIT. In some embodiments, the antibody of the present invention or its antigen-binding fragment binds to mammalian TIGIT, such as human TIGIT or cynomolgus monkey TIGIT.

[0150] Single-domain antibody

[0151] In some embodiments, the anti-TIGIT antibody of the present invention is a single-domain antibody, particularly a VHH antibody.

[0152] Single-domain antibodies or VHH antibodies have a molecular weight approximately one-tenth that of a human IgG molecule and a physical diameter of only a few nanometers. Due to their small molecular size, single-domain monoclonal antibodies have the following advantages compared to conventional four-chain antibodies: high stability and solubility, and the ability to recognize cryptic antigenic sites. In addition, single-domain antibodies are also less expensive to produce than conventional four-chain antibodies. In addition to being used as individual molecules, single-domain antibodies are also suitable components for constructing multispecific molecules.

[0153] In some embodiments, the single-domain antibodies of the present invention are VHH antibodies comprising or consisting of a heavy-chain variable region, which generally has the following structure: FR1-VHH CDR1-FR2-VHH CDR2-FR3-VHH CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4; VHH CDR1 to VHH CDR3 refer to complementary determining regions 1-3. The CDR sequences in the VHH variable region can be determined according to any CDR definition scheme described in the "Definitions" section, and preferably the boundaries of the three CDRs in the VHH sequence can be defined by IMGT.

[0154] In some embodiments, the anti-TIGIT VHH antibody of the present invention comprises

[0155] (i) the three complementary determining regions (CDRs) contained in the VH shown in any one of SEQ ID NOs: 1, 6, 9, 14, 16, 18, and 21, or

[0156] (ii) a sequence that contains a total of at least one and no more than 5, 4, 3, 2, or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three CDR regions relative to the sequence of (i);

[0157] Preferably, the CDR sequences are defined according to IMGT.

[0158] In some embodiments, the anti-TIGIT VHH antibody of the present invention comprises or consists of a heavy-chain variable region that contains

[0159] (i) the three complementary determining regions (CDRs) contained in the VH shown in any one of SEQ ID NOs: 1, 6, 9, 14, 16, 18, and 21, or

[0160] (ii) a sequence that contains a total of at least one and no more than 5, 4, 3, 2, or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three CDR regions relative to the sequence of (i);

[0161] Preferably, the CDR sequences are defined according to IMGT.

[0162] In some embodiments, the anti-TIGIT VHH antibody of the present invention comprises complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3. In some embodiments, the anti-TIGIT VHH of the present invention comprises or consists of a heavy chain variable region that comprises complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3.

[0163] In some embodiments, VHH CDR1 comprises or consists of an amino acid sequence selected from SEQ ID NO: 3, 8, or 11, or VHH CDR1 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO: 3, 8, or 11.

[0164] In some embodiments, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 4, 12, or 23, or VHH CDR2 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 4, 12, or 23.

[0165] In some embodiments, VHH CDR3 comprises or consists of an amino acid sequence selected from SEQ ID NO: 5 or 13, or VHH CDR3 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO: 5 or 13.

[0166] In some embodiments, the anti-TIGIT VHH antibody of the present invention comprises or consists of a heavy chain variable region that comprises

[0167] complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, wherein HCDR3 comprises or consists of an amino acid sequence selected from SEQ ID NO: 5 or 13, or HCDR3 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO: 5 or 13.

[0168] In some embodiments, the VHH CDR2 of the anti-TIGIT VHH antibody of the present invention comprises or consists of the following amino acid sequence:

[0169] ITTSXSA, preferably, X is selected from D or S (SEQ ID NO: 57).

[0170] In one embodiment, the anti-TIGIT VHH antibody of the present invention comprises complementarity determining regions (CDRs) VHHCDR1, VHH CDR2, and VHH CDR3, wherein

[0171] (i) VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4 or 23 or 57, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 5; or

[0172] (ii) VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 8, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4 or 23 or 57, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 5; or

[0173] (iii) VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 11, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 12, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 13.

[0174] In one embodiment, the anti-TIGIT VHH antibody of the present invention comprises or consists of a heavy chain variable region, wherein the heavy chain variable region comprises complementarity determining regions (CDRs) VHHCDR1, VHHCDR2, and VHHCDR3, wherein

[0175] (i) VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4 or 23 or 57, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 5; or

[0176] (ii) VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 8, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4 or 23 or 57, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 5; or

[0177] (iii) The VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 11, the VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 12, and the VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 13.

[0178] In some embodiments, the anti-TIGIT VHH antibody of the present invention comprises or consists of a heavy chain variable region, and the heavy chain variable region

[0179] (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in any one of SEQ ID NO: 1, 6, 9, 14, 16, 18 and 21; or

[0180] (ii) comprises or consists of the amino acid sequence shown in any one of SEQ ID NO: 1, 6, 9, 14, 16, 18 and 21; or

[0181] (iii) comprises an amino acid sequence having 1 or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence shown in any one of SEQ ID NO: 1, 6, 9, 14, 16, 18 and 21, and preferably, the amino acid alterations do not occur in the CDR regions.

[0182] In some embodiments, the anti-TIGIT VHH antibody of the present invention comprises or consists of the amino acid sequence shown in any one of SEQ ID NO: 1, 6, 9, 14, 16, 18 and 21.

[0183] In some embodiments, the VHH antibody of the present invention comprises CDR amino acid sequences and / or framework (FR) amino acid sequences derived from camelid heavy chain antibodies produced by immunizing camelids (such as alpacas). In some embodiments, the VHH monoclonal antibody of the present invention derived from camelid heavy chain antibodies can be engineered, for example, to comprise framework region sequences derived from human amino acid sequences (i.e., human antibodies) or other non-camelid mammalian species. In one embodiment, to further improve the properties (such as affinity) of the engineered antibody, camelid amino acid residues located at corresponding positions in the parental camelid antibody can be introduced into the engineered antibody at one or more positions (such as framework regions) by back mutation.

[0184] In one embodiment, the VHH antibody of the present invention is a chimeric antibody.

[0185] In one embodiment, the VHH antibody of the present invention is a humanized antibody. Humanization can be achieved by replacing one or more amino acid residues, especially framework region sequences, in a non-human native VHH sequence (such as a VHH sequence from a camelid or alpaca immunization) with residues at the corresponding positions of the heavy chain VH of a conventional human antibody. Methods for humanizing VHH are well known in the art, such as the method described in Example 3. Generally, the humanization replacements are made in a manner that maintains the favorable binding properties of the single-domain antibody. Assays for determining the biological properties of humanized single-domain antibodies, such as binding affinity, are well known in the art to identify and select suitable humanization residue mutations or combinations of mutations.

[0186] In some embodiments, the humanized single-domain antibodies of the present invention can be obtained by a method comprising the following steps:

[0187] ① Determine the CDR loop structures of the parental single-domain antibody (such as a camelid VHH antibody screened from a phage display library);

[0188] ② Find the closest homologous sequences as templates for each V / J region in the human germline sequence database, for example, by aligning the IMGT human heavy chain variable region germline gene database (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi), and select the heavy chain variable region germline genes with high homology to the VHH antibody as templates;

[0189] ③ Transplant the CDRs of the VHH antibody into the selected corresponding human templates respectively to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CR3-FR4. Preferably, the framework sequence used for replacement has a structural similarity to the framework sequence of the antibody to be humanized, for example, having a sequence identity of at least 80%, 85%, 90%, or more than 95%, 96%, 97%, 98%, 99%;

[0190] ④ Optionally, revert the key amino acids in the FR region to the corresponding amino acids of the nanobody (VHH antibody) to ensure the original affinity, that is, obtain the humanized anti-TIGIT VHH antibody, and optionally sequence the VHH antibody.

[0191]

[0192] ​In some embodiments, the reverse mutation site is selected from one or more of T28, L78, W103, R83, V37, G44, L45, and W47. In some embodiments, the reverse mutation is selected from one or more of T28P; L78V; W103K; R83K; V37F; G44E; L45R; W47F. In some embodiments, the combination of sites of reverse mutations in the humanized VHH antibody is selected from:

[0193] (1) T28, L78, and W103;

[0194] (2) L78, R83, and W103; or

[0195] (3) V37, G44, L45, and W47.

[0196] In some embodiments, the combination of reverse mutations in the humanized VHH antibody is selected from:

[0197] (1) T28P, L78V, and W103K (e.g., for heavy chain template IGHJ4*01);

[0198] (2) L78V, R83K, and W103K (e.g., for heavy chain template IGHV3-48*01); or

[0199] (3) V37F, G44E, L45R, and W47F (e.g., for heavy chain template IGHV3-23*01).

[0200] In some embodiments, the germline genes of the heavy chain variable region applicable to the humanization of the VHH antibody of the present invention are selected from IGHJ4*01, IGHV3-48*01, or IGHV3-23*01.

[0201] In some embodiments, the present invention also provides functional variants of the single-domain antibodies (particularly VHH antibodies) of the present invention. The functional variants can be obtained by introducing mutations into the coding nucleic acid sequences of the exemplary single-domain antibodies of the present invention, such as into the CDR sequences and / or FR sequences, by methods well-known in the art, such as random or site-directed mutagenesis, and then screening (e.g., by screening a phage display library) for variants that retain the desired properties. Generally, the functional variants retain significant sequence identity with the parental single-domain antibody (or VHH). Preferably, the functional variants retain the desired biological properties of the parental single-domain antibody (or VHH), e.g., the variant has comparable (e.g., at least 50%, 60%, 70%, 80%, preferably more than 90%) biological activity or improved biological activity (e.g., 110 - 150% or higher) relative to the biological activity of the parent. The desired biological properties include, for example, but are not limited to, binding affinity for the target antigen (e.g., CD155) (measured by the KD value), activity of blocking the binding of the target antigen to the receptor (e.g., measured by the IC50 value), activity of activating T cells in in vitro or in vivo experiments (e.g., measured by the amount of cytokines released), and inhibiting tumor growth / survival in in vitro or in vivo experiments.

[0202] In some embodiments, the present invention provides affinity variants of the VHH polypeptides of the present invention. Preferably, the affinity variants exhibit one or more amino acid changes in the amino acid sequence relative to their source parental single-domain antibody, wherein the affinity variant has an altered binding affinity for the target antigen compared to the parental antibody.

[0203] In some embodiments, the antibody can also be engineered for stability, e.g., mutating asparagine in the antibody to eliminate deamidation. In one embodiment, the CDR2 of the VHH antibody of the present invention contains the D63S mutation to eliminate deamidation.

[0204] Heavy-chain antibody

[0205] In another aspect of the present invention, the present invention also provides heavy-chain antibodies comprising the heavy-chain variable region of the VHH antibody of the present invention.

[0206] In some embodiments, the single-domain antibodies or VHHs of the present invention (e.g., camelid VHHs or their humanized forms) can be linked to the constant region of a human antibody or a portion thereof, such as the Fc region, to generate a heavy-chain antibody comprising a VHH-constant region or VHH-CH1-Fc or VHH-Fc. In one embodiment, the heavy-chain antibody comprises the VHH antibody of the present invention and an Fc region at its C-terminus. In some embodiments, the VHH is linked to the Fc via a hinge region or a portion thereof, such as a hinge region from IgG (e.g., the hinge region of IgG1, 2, 3, or 4) or a portion thereof.

[0207] In some embodiments, the anti-TIGIT heavy-chain antibody of the present invention comprises a VHH or the heavy-chain variable region thereof as defined herein, and a heavy-chain constant region or the Fc region of the heavy-chain constant region. In some embodiments, a linker peptide is included between the VHH or its heavy-chain variable region and the heavy-chain constant region or the Fc region, such as an antibody hinge region or a portion thereof, such as a hinge region from IgG or a portion thereof (including native or mutated IgG hinge regions or portions thereof).

[0208] In some embodiments, the linker peptide is a hinge region or a portion thereof from human IgG1, 2, 3, or 4, including native or mutated hinge regions or portions thereof, such as from the human IgG1 hinge region, such as the linker peptide is EPKSS (SEQ ID NO: 43).

[0209] In one embodiment, the heavy-chain antibody comprises an Fc portion from a camelid (e.g., alpaca). In one embodiment, the heavy-chain antibody is produced and isolated by immunizing the camelid, such as an alpaca. A variety of methods are known in the art for immunizing camelids and isolating the generated VHH antibodies or heavy-chain antibodies specific for the antigen of interest.

[0210] In some embodiments, the heavy-chain antibody comprises a constant region from a human or non-human primate (e.g., cynomolgus monkey) antibody, such as a constant region from human IgG1, human IgG2, human IgG3, or human IgG4.

[0211] In some embodiments, the heavy-chain antibody comprises an Fc portion from a human or non-human primate (e.g., cynomolgus monkey). In a further embodiment, the heavy-chain antibody comprises a human IgG Fc region, such as a human IgG1, human IgG2, human IgG3, or human IgG4 Fc region, preferably a human IgG1 or human IgG4 Fc region, such as a human IgG1 Fc region.

[0212] In one embodiment, the heavy chain antibody according to the present invention can dimerize through the Fc region with another polypeptide chain containing the Fc region (such as another heavy chain antibody that is the same or different). Thus, in one embodiment, the present invention also provides a homologous or heterologous multimeric protein comprising the heavy chain antibody of the present invention. In a preferred embodiment, it is preferred that the protein comprises a heavy chain antibody formed by the pairing of two identical heavy chain antibody chains.

[0213] The Fc region of the present invention can be mutated to obtain desired properties. Mutations of the Fc region are known in the art. In one embodiment, the Fc region is modified in terms of the properties of its effector functions. In one embodiment, the effector function has been enhanced relative to the wild-type isotype Fc region. In one embodiment, the effector function of the Fc region is improved by mutating the Fc region, such as ADCC, for example, by mutating at one or more of the following sites: S239, A330, and I332 (according to EU numbering). In one embodiment, ADCC is improved by mutations in the following combinations of sites: S239, A330, and I332 (according to EU numbering). In one embodiment, the mutations are selected from 1, 2, or 3 of S239D, A330L, and I332E. In one embodiment, the mutations that alter the effector function are the following mutation combination: S239D, A330L, and I332E (reference: "Engineered antibody Fc variants with enhanced effector function. Proc Natl Acad Sci USA. 2006 Mar 14;103(11):4005 - 10.").

[0214] In some embodiments, the Fc region is the Fc region from IgG1, which contains the mutations S239D, A330L, and I332E (according to EU numbering).

[0215] In some embodiments, the Fc region:

[0216] (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 40, 41, or 42; or

[0217] (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 40, 41, or 42; or

[0218] (iii) an amino acid sequence having 1 or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 40, 41 or 42.

[0219] In some embodiments, the Fc region is from IgG1 and comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO: 40 or 41.

[0220] In some embodiments, the Fc region is from IgG1 and comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO: 41 and comprises the following combination of mutations: S239D, A330L and I332E (EU numbering).

[0221] In some embodiments, the Fc region is from IgG4 and comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO: 42.

[0222] In some embodiments, the antibody TIGIT antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain, which comprises a heavy chain variable region, an Fc region, and a linker peptide connecting the heavy chain variable region and the Fc region. Preferably, the linker peptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 43.

[0223] In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof of the present invention comprises or consists of a heavy chain, which comprises the heavy chain variable region of the VHH of the present invention, a linker peptide and an Fc region or consists of the foregoing, wherein the heavy chain

[0224] (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence selected from any one of SEQ ID NO: 2, 7, 10, 15, 17, 19, 20 or 22; or

[0225] (ii) comprising or consisting of an amino acid sequence shown in any one of SEQ ID NO: 2, 7, 10, 15, 17, 19, 20 or 22; or

[0226] (iii) comprising an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence shown in any one of SEQ ID NO: 2, 7, 10, 15, 17, 19, 20 or 22, preferably, said amino acid alterations do not occur in the CDR regions.

[0227] Bispecific antibody or multispecific antibody

[0228] In certain embodiments, the present invention also encompasses multispecific molecules comprising the VHH or heavy chain antibody or fragments thereof of the present invention, such as bispecific antibodies. The multispecific antibody molecule can be, for example, a trispecific antibody molecule, which comprises a first binding specificity against TIGIT and second and third binding specificities against one or more molecules.

[0229] Thus, another aspect of the present invention relates to a bispecific antibody, which comprises

[0230] a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds to TIGIT, and the second antigen-binding region specifically binds to a tumor-associated antigen or an immune checkpoint molecule, such as PD-1, PD-L1 or PD-L2 or CTLA4.

[0231] In some embodiments, the first antigen-binding region comprises the anti-TIGTI VHH antibody or heavy chain antibody described herein, particularly the VHH antibody. Preferably, the first antigen-binding region comprises or consists of the anti-TIGIT VHH of the present invention, and more preferably the VHH is a humanized VHH.

[0232] In some embodiments, the second antigen-binding region comprises the PD1 antibody from WO2019219064A or its antigen-binding fragment or domain, such as a fragment or domain comprising the PD1 antibody of WO2019219064A. The second antigen-binding region of the bispecific antibody applicable to the present invention can comprise or consist of a full-length anti-PD-1 antibody or its antigen-binding fragment, as long as it can specifically bind to PD-1, including but not limited to, for example, a full-length antibody specifically binding to PD-1, a single-chain Fv, a Fab, a Fab', a (Fab)2, a single-domain antibody, a VHH or a heavy chain antibody, etc.

[0233] In some embodiments, the second antigen-binding region comprises from the Ipilimumab antibody or an antigen-binding fragment or domain thereof, such as a fragment or domain comprising the Ipilimumab antibody. The second antigen-binding region of the bispecific antibody suitable for the present invention may comprise, consist of, or be composed of an anti-CTLA-4 full-length antibody or an antigen-binding fragment thereof, provided that it is capable of specifically binding to CTLA-4, including but not limited to, for example, a full-length antibody specifically binding to CTLA-4, single-chain Fv, Fab, Fab', (Fab)2, single-domain antibody, VHH, or heavy-chain antibody, etc.

[0234] In the bispecific antibody according to the present invention, the anti-TIGIT VHH of the present invention as the first antigen-binding region can be linked to the N-terminus or C-terminus of the second antigen-binding region, such as linked to the C-terminus of the Fc fragment of the second antigen-binding region, or linked to the N-terminus of the VH fragment of the heavy chain of the second antigen-binding region, or can be inserted between the Fab fragment (Fab fragment heavy chain) and the Fc fragment of the second antigen-binding region, that is, linked to the C-terminus of the Fab fragment heavy chain and the N-terminus of the Fc fragment. In some embodiments, the first and second antigen-binding regions are linked by a linker (such as when the anti-TIGIT VHH is inserted between the Fab fragment and the Fc fragment of the second antigen antibody). In one embodiment, the linker is a peptide having a length of about 3 to about 20 amino acids. Preferably, the linker comprises the (GGS)n amino acid sequence, where n is an integer of 1, 2, 3, 4, or 5, preferably n = 1.

[0235] In some embodiments, the bispecific antibody of the present invention has the following structure:

[0236] Heavy chain: from the N-terminus to the C-terminus, the heavy chain variable region VH of the second antigen antibody - heavy chain constant region CH1 - heavy chain constant region Fc - anti-TIGIT VHH; or

[0237] from the N-terminus to the C-terminus, the heavy chain variable region VH of the second antigen antibody - heavy chain constant region CH1 - anti-TIGIT VHH - heavy chain constant region Fc; or

[0238] from the N-terminus to the C-terminus, anti-TIGIT VHH - the heavy chain variable region VH of the second antigen antibody - heavy chain constant region CH1 - heavy chain constant region Fc;

[0239] Light chain: from the N-terminus to the C-terminus, the light chain variable region of the second antigen antibody - light chain constant region CL.

[0240] In some embodiments, the second antigen is PD-1, such as human PD-1. In some embodiments, the second antigen is CTLA-4, such as human CTLA-4.

[0241] In some embodiments, the bispecific antibody of the present invention has two heavy chains and two light chains, preferably two identical heavy chains and two light chains.

[0242] Preferably, the structure of the bispecific antibody is as Figure 6A or Figure 6B or Figure 14A shown.

[0243] In other embodiments, the anti-TIGIT single-domain antibody VHH is linked to the heavy chain constant region Fc portion at the N-terminus or C-terminus via a linker. In one embodiment, the linker is a peptide about 3 to about 20 amino acids in length. Preferably, the linker comprises the (GGS)n amino acid sequence, where n is an integer of 1, 2, 3, 4, or 5, preferably n = 1.

[0244] In the bispecific antibody according to the present invention, the anti-TIGIT VHH of the present invention, as the first antigen-binding region, can combine with Fc to form a heavy-chain antibody and heterodimerize with the second antigen-binding region to form a bispecific antibody.

[0245] In some embodiments, the bispecific antibody of the present invention has the following structure:

[0246] Heavy chain 1: From the N-terminus to the C-terminus, the heavy chain variable region VH of the second antigen antibody - heavy chain constant region CH1 - heavy chain constant region Fc;

[0247] Heavy chain 2: One or more (e.g., 2) tandem anti-TIGIT VHH - heavy chain constant region Fc

[0248] Light chain: From the N-terminus to the C-terminus, the light chain variable region of the second antigen antibody - light chain constant region CL.

[0249] In some embodiments, the bispecific antibody of the present invention has one heavy chain 1, one heavy chain 2, and one light chain.

[0250] Preferably, the structure of the bispecific antibody is as Figure 14B or shown in 14C.

[0251] In some embodiments, the anti-TIGIT single-domain antibody VHH is linked to the N-terminus of the heavy chain constant region Fc via a linking peptide. In some embodiments, the linking peptide is the hinge region or a portion thereof from human IgG1, 2, 3, or 4, including the native or mutated hinge region or a portion thereof, such as from the human IgG1 hinge region, for example, the linking peptide is EPKSS (SEQ ID NO: 43).

[0252] In some embodiments, when the heavy chain 2 comprises multiple tandem anti-TIGIT single domain antibodies VHH, the individual tandem VHHs may be linked by a linker. In one embodiment, the linker is a peptide having a length of from about 3 to about 20 amino acids. Preferably, the linker comprises the (GGGGS)n amino acid sequence, where n is an integer of 1, 2, 3, 4, or 5, preferably n = 1.

[0253] In some embodiments, the second antigen is CTLA-4, such as human CTLA-4.

[0254] In one embodiment of the invention, the anti-TIGIT VHH of the invention is defined as described above.

[0255] In one embodiment of the invention, the heavy chain variable region and / or the light chain variable region of the anti-PD1 antibody in the bispecific antibody of the invention is derived from the PD1 antibody of WO2019219064A.

[0256] In some embodiments, the heavy chain variable region VH of the anti-PD1 antibody of the invention comprises three complementarity determining regions VHCDR1, VHCDR2, and VHCDR3. In some embodiments, the light chain variable region of the anti-PD1 antibody of the invention comprises three complementarity determining regions VLCDR1, VLCDR2, and VLCDR3.

[0257] In some embodiments, the complementarity determining regions VHCDR1, VHCDR2, and VHCDR3 of the three heavy chain variable regions of the invention are derived from a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO:24 or consisting of said amino acid sequence. Preferably, the CDRs are defined by Kabat.

[0258] In some embodiments, the complementarity determining regions VLCDR1, VLCDR2, and VLCDR3 of the three light chain variable regions of the invention are derived from a light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO:34 or consisting of said amino acid sequence. Preferably, the CDRs are defined by Kabat.

[0259] In some embodiments, the complementarity determining region VHCDR1 of the anti-PD1 antibody of the invention comprises the amino acid sequence of SEQ ID NO:25 or consists of the same.

[0260] In some embodiments, the complementarity determining region VHCDR2 of the anti-PD1 antibody of the invention comprises the amino acid sequence of SEQ ID NO:26 or consists of the same.

[0261] In some embodiments, the complementarity determining region VHCDR3 of the anti-PD1 antibody of the invention comprises the amino acid sequence of SEQ ID NO:27 or consists of the same.

[0262] In some embodiments, the variable light chain complementary determining region VLCDR1 of the anti-PD1 antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 35.

[0263] In some embodiments, the variable light chain complementary determining region VLCDR2 of the anti-PD1 antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 36.

[0264] In some embodiments, the variable light chain complementary determining region VLCDR3 of the anti-PD1 antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 37.

[0265] In some embodiments, the variable heavy chain VH of the anti-PD1 antibody comprises VHCDR1, VHCDR2 and VHCDR3, wherein VHCDR1 comprises or consists of the sequence shown in SEQ ID NO: 25; VHCDR2 comprises or consists of the sequence shown in SEQ ID NO: 26; and / or VHCDR3 comprises or consists of the sequence shown in SEQ ID NO: 27.

[0266] In some embodiments, the variable light chain VL of the anti-PD1 antibody comprises VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 comprises or consists of the sequence shown in SEQ ID NO: 35; VLCDR2 comprises or consists of the sequence shown in SEQ ID NO: 36; and / or VLCDR3 comprises or consists of the sequence shown in SEQ ID NO: 37.

[0267] In some embodiments, the variable heavy chain VH of the anti-PD1 antibody of the present invention comprises the amino acid sequence set forth in SEQ ID NO: 24, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 24, or consists of the amino acids set forth in SEQ ID NO: 24. In some embodiments, the variable heavy chain VH comprises an amino acid sequence having one or several (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID NO: 24, said mutations being, for example, substitutions, deletions or additions, preferably substitutions, such as conservative substitutions. In some preferred embodiments, said mutations are not present in the CDRs, such as VHCDR1, VHCDR2 or VHCDR3.

[0268] In some embodiments, the variable light chain region VL of the anti-PD1 antibody of the present invention comprises the amino acid sequence set forth in SEQ ID NO: 34, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 34, or consists of the amino acids set forth in SEQ ID NO: 34. In some embodiments, the variable light chain region VL comprises an amino acid sequence having one or several (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID NO: 34, said mutations being, for example, substitutions, deletions or additions, preferably substitutions, such as conservative substitutions. In some preferred embodiments, said mutations are not present in the CDRs, such as VLCDR1, VLCDR2 or VLCDR3 of VL.

[0269] In one embodiment of the present invention, the variable heavy chain region and / or the variable light chain region of the anti-CTLA-4 antibody in the bispecific antibody of the present invention are from Ipilimumab.

[0270] In some embodiments, the variable heavy chain region VH of the anti-CTLA-4 antibody of the present invention comprises three complementarity determining regions VHCDR1, VHCDR2 and VHCDR3. In some embodiments, the variable light chain region of the anti-CTLA-4 antibody of the present invention comprises three complementarity determining regions VLCDR1, VLCDR2 and VLCDR3.

[0271] In some embodiments, the complementarity determining regions VHCDR1, VHCDR2 and VHCDR3 of the three variable heavy chain regions of the present invention are from a variable heavy chain region VH comprising the amino acid sequence set forth in SEQ ID NO: 62 or consisting of said amino acid sequence, preferably, said CDRs are defined by Kabat.

[0272] In some embodiments, the complementarity determining regions VLCDR1, VLCDR2 and VLCDR3 of the three variable light chain regions of the present invention are from a variable light chain region VL comprising the amino acid sequence set forth in SEQ ID NO: 67 or consisting of said amino acid sequence, preferably, said CDRs are defined by Kabat.

[0273] In some embodiments, the complementarity determining region VHCDR1 of the anti-CTLA-4 antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 59.

[0274] In some embodiments, the complementarity determining region VHCDR2 of the anti-CTLA-4 antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 60.

[0275] In some embodiments, the complementarity determining region VHCDR3 of the anti-CTLA-4 antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 61.

[0276] In some embodiments, the complementarity determining region VLCDR1 of the anti-CTLA-4 antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 64.

[0277] In some embodiments, the complementarity determining region VLCDR2 of the anti-CTLA-4 antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 65.

[0278] In some embodiments, the complementarity determining region VLCDR3 of the anti-CTLA-4 antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 66.

[0279] In some embodiments, the heavy chain variable region VH of the anti-CTLA-4 antibody comprises VHCDR1, VHCDR2 and VHCDR3, wherein VHCDR1 comprises or consists of the sequence shown in SEQ ID NO: 59; VHCDR2 comprises or consists of the sequence shown in SEQ ID NO: 60; and / or VHCDR3 comprises or consists of the sequence shown in SEQ ID NO: 61.

[0280] In some embodiments, the light chain variable region VL of the anti-CTLA-4 antibody comprises VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 comprises or consists of the sequence shown in SEQ ID NO: 64; VLCDR2 comprises or consists of the sequence shown in SEQ ID NO: 65; and / or VLCDR3 comprises or consists of the sequence shown in SEQ ID NO: 66.

[0281] In some embodiments, the heavy chain variable region VH of the anti-CTLA-4 antibody of the present invention comprises the amino acid sequence set forth in SEQ ID NO: 62, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 62, or consists of the amino acids set forth in SEQ ID NO: 62. In some embodiments, the heavy chain variable region VH comprises an amino acid sequence having one or several (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID NO: 62, said mutations being, for example, substitutions, deletions or additions, preferably substitutions, such as conservative substitutions. In some preferred embodiments, said mutations are not present in the CDRs, such as VHCDR1, VHCDR2 or VHCDR3.

[0282] In some embodiments, the light chain variable region VL of the anti-CTLA-4 antibody of the present invention comprises the amino acid sequence set forth in SEQ ID NO: 67, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 67, or consists of the amino acids set forth in SEQ ID NO: 67. In some embodiments, the light chain variable region VL comprises an amino acid sequence having one or several (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID NO: 67, said mutations being, for example, substitutions, deletions or additions, preferably substitutions, such as conservative substitutions. In some preferred embodiments, said mutations are not present in the CDRs, such as VLCDR1, VLCDR2 or VLCDR3.

[0283] In some embodiments, the heavy chain constant region CH1 of the second antigen antibody in the bispecific antibody of the present invention is from IgG, such as IgG1, IgG2, IgG3 or IgG4. Preferably, the heavy chain constant region CH1 is from IgG1 or IgG4.

[0284] In some embodiments, the heavy chain constant region CH1

[0285] (i) comprises the amino acid sequence set forth in SEQ ID NO: 28 or 31, or

[0286] (ii) It is from IgG1 and contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 28, or it is from IgG4 and contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 31, or

[0287] (iii) Composed of the amino acids set forth in SEQ ID NO: 28 or 31; or

[0288] (iv) An amino acid sequence having one or several (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID NO: 28 or 31, such as substitution, deletion or addition, preferably substitution, such as conservative substitution.

[0289] In some embodiments, the CH1 and Fc regions of the heavy chain constant region in the bispecific antibody of the present invention are from the same IgG, such as IgG1, IgG2, IgG3 or IgG4, preferably both from IgG1 or IgG4.

[0290] In some embodiments, the light chain constant region CL of the bispecific antibody of the present invention is a Lambda or Kappa light chain constant region, preferably the Kappa light chain constant region. In some embodiments, the light chain constant region CL

[0291] (i) Contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence selected from SEQ ID NO: 38 or is composed of said amino acid sequence;

[0292] (ii) Contains the amino acid sequence selected from SEQ ID NO: 38 or is composed of said amino acid sequence; or

[0293] (iii) Contains an amino acid sequence having 1 or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitution, more preferably amino acid conservative substitution) compared to the amino acid sequence selected from SEQ ID NO: 38 or is composed of said amino acid sequence.

[0294] In some embodiments, the Fc region of the heavy chain antibody applicable to the present invention is also applicable to the bispecific antibody of the present invention.

[0295] In some embodiments, the heavy chain constant region Fc in the bispecific antibody of the present invention is from IgG, such as IgG1, IgG2, IgG3 or IgG4. In some embodiments, the Fc region is from IgG1 or from IgG4.

[0296] In one embodiment, the two Fc regions in the bispecific antibody of the present invention dimerize to form a dimeric Fc.

[0297] In some embodiments, for example, when the bispecific antibody comprises the same heavy chain, the first and second Fc regions are the same. In other embodiments, for example, when the bispecific antibody comprises different heavy chains, the first Fc region and the second Fc region are different, and the two pair and heterodimerize.

[0298] The Fc region fragment applicable to the antibody molecule of the present invention can be any antibody Fc region. The Fc region can include a native sequence Fc region and a variant Fc region. The native sequence Fc domain encompasses various naturally occurring immunoglobulin Fc sequences, such as the Fc regions of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520.). For example, the Fc region of the antibody of the present invention can comprise two or three constant domains, namely the CH2 domain, the CH3 domain and optionally the CH4 domain. In some embodiments, the antibody Fc region can also carry an IgG hinge region or a partial IgG hinge region at the N-terminus, for example, the IgG1 hinge region or a partial IgG1 hinge region. Mutations can be contained in the hinge region. In some embodiments, the hinge region can be EPKSS or EPKSC.

[0299] Preferably, the Fc region of the antibody of the present invention comprises, from the N-terminus to the C-terminus: CH2-CH3, or comprises, from the N-terminus to the C-terminus: hinge region-CH2-CH3. In some embodiments, the Fc region applicable to the antibody or bispecific antibody of the present invention is a human IgG Fc, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 or human IgG4 Fc. In some embodiments, the Fc region:

[0300] (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO: 40 or 42; or

[0301] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 40 or 42; or

[0302] (iii) comprising an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared with the amino acid sequence shown in SEQ ID NO: 40 or 42.

[0303] The Fc region of the present invention can be mutated to obtain desired properties. Mutations of the Fc region are known in the art. In one embodiment, the Fc region is modified in terms of the properties of its effector functions. In one embodiment, the effector function has been enhanced relative to the wild-type isotype Fc region. In one embodiment, the effector function of the Fc region is improved by mutating the Fc region, such as ADCC, for example by mutating at one or more of the following sites: S239, A330, and I332 (according to EU numbering). In one embodiment, ADCC is improved by mutations in the following combinations of sites: S239, A330, and I332 (according to EU numbering). In one embodiment, the mutations are selected from 1, 2, or 3 of S239D, A330L, and I332E. In one embodiment, the mutations that alter the effector function are the following mutation combination: S239D, A330L, and I332E.

[0304] As understood by those skilled in the art, in order to facilitate the formation of the bispecific antibody of the present invention as a heterodimer, the Fc region contained in the bispecific antibody of the present invention may contain mutations that facilitate heterodimerization. In one embodiment, mutations are introduced into the CH3 region of the two Fc regions.

[0305] Methods for promoting Fc region heterodimerization are known in the art. For example, the CH3 region of the first Fc region and the CH3 region of the second Fc region are engineered in a complementary manner such that each CH3 region (or the heavy chain containing it) can no longer homodimerize with itself but is forced to heterodimerize with the other complementary engineered CH3 region (such that the first and second CH3 regions heterodimerize and no homodimers are formed between two first CH3 regions or two second CH3 regions).

[0306] Preferably, based on the Knob-in-Hole technology, corresponding knob mutations and Hole mutations are introduced into the first monomeric Fc region and the second monomeric Fc region, respectively. For this technology, see, for example, Merchant, A.M., et al. (1998). "An efficient route to human bispecific IgG." Nat Biotechnol 16(7):677-681.

[0307] In a particular embodiment, in the CH3 region of one Fc region, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) (knob mutation); while in the CH3 region of the other Fc region, the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (Hole mutation), optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the EU index).

[0308] In yet another embodiment, in the CH3 region of one Fc region, the knob mutation comprises or consists of: the threonine residue at position 366 is replaced with a tryptophan residue (T366W) and the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamate residue at position 356 is replaced with a cysteine residue (E356C) (in particular, the serine residue at position 354 is replaced with a cysteine residue); while in the CH3 region of the other Fc region, the Hole mutation comprises or consists of: the tyrosine residue at position 407 is replaced with a valine residue (Y407V), optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the EU index), optionally the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C) (numbering according to the EU index).

[0309] In a specific embodiment, one Fc region comprises the amino acid substitutions S354C and T366W (knob mutation), and the other Fc region comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (Hole mutation) (numbering according to the EU index).

[0310] Thus, in a specific embodiment, the two Fc regions of the bispecific antibody of the present invention heterodimerize, wherein

[0311] a) one Fc-region polypeptide comprises the mutation T366W, while the other Fc-region polypeptide comprises the mutation

[0312] T366S, L368A, and Y407V, or

[0313] b) One Fc-region polypeptide contains the mutations T366W and Y349C, while the other Fc-region polypeptide contains the mutations T366S, L368A, Y407V, and S354C, or

[0314] c) One Fc-region polypeptide contains the mutations T366W and S354C, while the other Fc-region polypeptide contains the mutations T366S, L368A, Y407V, and Y349C.

[0315] In some embodiments, the Fc region further contains other mutations that facilitate the purification of the heterodimer. For example, the H435R mutation (Eric J. Smith,, Scientific Reports|5:17943|DOI:10.1038 / srep17943) can be introduced into one of the Fc regions of the heterodimer (e.g., the Fc region with the Hole mutation) to facilitate the purification of the heterodimer using Protein A. In other embodiments, for the heterodimer monomers containing a hinge region, mutations can also be introduced into the hinge region, such as C220S, to facilitate the formation of the heterodimer.

[0316] In a specific embodiment, the two Fc regions of the bispecific antibody of the present invention heterodimerize, wherein

[0317] The first Fc region contains a knot mutation, which contains the amino acid sequence SEQ ID NO:54 or an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or higher identity, with it or consisting of it; in some embodiments, the Fc region contains an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or higher identity, with SEQ ID NO:54 and contains a knot mutation (e.g., S354C and T366W); in some embodiments, the Fc region contains or does not contain the hinge region EPKSS or EPKSC;

[0318] The second Fc region contains a button mutation, which contains the amino acid sequence SEQ ID NO:75 and an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or higher identity, with it or consisting of it. In some embodiments, the Fc region contains an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or higher identity, with SEQ ID NO:75 and contains a button mutation; in some embodiments, the Fc region contains or does not contain the hinge region EPKSS or EPKSC.

[0319] In a specific embodiment, the two Fc regions of the bispecific antibody of the present invention heterodimerize, wherein

[0320] The first Fc region contains a knot mutation, which contains the amino acid sequence SEQ ID NO:78 or an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or higher identity with it or consists of it; in some embodiments, the Fc region contains an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or higher identity with SEQ ID NO:78 and contains S239D, A330L and I332E mutations and a knot mutation (such as S354C and T366W); in some embodiments, the Fc region contains or does not contain the hinge region EPKSS or EPKSC;

[0321] The second Fc region contains a buckle mutation, which contains the amino acid sequence SEQ ID NO:77 or an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or higher identity with it or consists of it. In some embodiments, the Fc region contains an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or higher identity with SEQ ID NO:77 and contains S239D, A330L and I332E mutations and a buckle mutation; in some embodiments, the Fc region contains or does not contain the hinge region EPKSS or EPKSC.

[0322] In some embodiments, the anti-TIGIT single-domain antibody VHH in the bispecific antibody of the present invention contains HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 are from the VHH shown in SEQ ID NO:18 or 21. In some embodiments, the HCDR1 contains the amino acid sequence shown in SEQ ID NO:3 or consists of it, the HCDR2 contains the amino acid sequence shown in SEQ ID NO:4 or 23 or 57 or consists of it, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:5 or consists of it. In some embodiments, the anti-TIGIT single-domain antibody VHH in the bispecific antibody of the present invention contains a heavy chain variable region VH or consists of a heavy chain variable region VH, and the heavy chain variable region VH contains the amino acid sequence shown in SEQ ID NO:18 or 21 or consists of the amino acid sequence.

[0323] In some embodiments, the bispecific antibody of the present invention contains

[0324] Heavy chain: from the N-terminus to the C-terminus, the heavy chain variable region VH of the second antigen antibody - the heavy chain constant region CH1 - the heavy chain constant region Fc - the anti-TIGIT VHH; or

[0325] From the N-terminus to the C-terminus, the heavy chain variable region VH - heavy chain constant region CH1 - anti-TIGIT VHH - heavy chain constant region Fc of the second antigen antibody

[0326] Light chain: From the N-terminus to the C-terminus, the light chain variable region - light chain constant region CL of the second antigen antibody,

[0327] wherein the second antigen is PD-1;

[0328] wherein the heavy chain

[0329] (i) comprises the amino acid sequence of SEQ ID NO: 30, 32 or 33, or

[0330] (ii) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence described in SEQ ID NO: 30, 32 or 33, or

[0331] (iii) consists of the amino acids described in SEQ ID NO: 30, 32 or 33, or

[0332] (iv) comprises an amino acid sequence having one or several (preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared with the amino acid sequence shown in SEQ ID NO: 30, 32 or 33, said mutations being, for example, substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, preferably, said mutations do not exist in the CDRs of anti-TIGIT VHH, or do not exist in the VHH of anti-TIGIT, or do not exist in the CDRs of the heavy chain variable region of the anti-PD1 antibody, or preferably do not exist in the heavy chain variable region;

[0333] and / or

[0334] light chain

[0335] (i) comprises the amino acid sequence of SEQ ID NO: 39, or

[0336] (ii) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence described in SEQ ID NO: 39, or

[0337] (iii) consists of the amino acids described in SEQ ID NO: 39, or

[0338] (iv) comprising an amino acid sequence having one or several (preferably not exceeding 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared with the amino acid sequence shown in SEQ ID NO: 39, said mutations being, for example, substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, preferably, said mutations are not present in the CDRs of the light chain variable region of the anti-PD1 antibody, or preferably are not present in the light chain variable region.

[0339] In some embodiments, the bispecific antibody of the present invention comprises

[0340] (1) a heavy chain, which comprises the amino acid sequence of SEQ ID NO: 30, 32 or 33, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 30, 32 or 33, or consists of said sequence; and

[0341] (2) a light chain, which comprises the amino acid sequence of SEQ ID NO: 39, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 39.

[0342] In some embodiments, the bispecific antibody of the present invention comprises

[0343] Heavy chain: from the N-terminus to the C-terminus, the heavy chain variable region VH of the anti-TIGIT VHH-second antigen antibody - the heavy chain constant region CH1 - the heavy chain constant region Fc;

[0344] Light chain: from the N-terminus to the C-terminus, the light chain variable region of the second antigen antibody - the light chain constant region CL,

[0345] wherein the second antigen is CTLA-4,

[0346] wherein the heavy chain

[0347] (i) comprises the amino acid sequence of SEQ ID NO: 69, or

[0348] (ii) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 69, or

[0349] (iii) consists of the amino acids of SEQ ID NO: 69, or

[0350] (iv) comprising an amino acid sequence having one or several (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID NO: 69, said mutations being, for example, substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, preferably, said mutations are not present in the CDRs of the anti-TIGIT VHH, or not present in the VHH against TIGIT, or not present in the CDRs of the heavy chain variable region of the anti-CTLA-4 antibody, or preferably not present in the heavy chain variable region;

[0351] and / or

[0352] light chain

[0353] (i) comprising the amino acid sequence of SEQ ID NO: 68, or

[0354] (ii) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 68, or

[0355] (iii) consisting of the amino acids set forth in SEQ ID NO: 68, or

[0356] (iv) comprising an amino acid sequence having one or several (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID NO: 68, said mutations being, for example, substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, preferably, said mutations are not present in the CDRs of the light chain variable region of the anti-CTLA-4 antibody, or preferably not present in the light chain variable region.

[0357] In some embodiments, the bispecific antibody of the invention comprises

[0358] (1) a heavy chain, which comprises the amino acid sequence of SEQ ID NO: 69, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 69, or consists of said sequence; and

[0359] (2) a light chain, which comprises the amino acid sequence of SEQ ID NO: 68, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 68.

[0360] In some embodiments, the bispecific antibody of the invention comprises

[0361] Heavy chain 1: from the N-terminus to the C-terminus, variable heavy chain region VH of the second antigen antibody - constant heavy chain region CH1 - constant heavy chain region Fc;

[0362] Heavy chain 2: one or more (e.g., two) tandem anti-TIGIT VHH - constant heavy chain region Fc

[0363] Light chain: from the N-terminus to the C-terminus, variable light chain region of the second antigen antibody - constant light chain region CL,

[0364] wherein the second antigen is CTLA-4,

[0365] wherein heavy chain 1

[0366] (i) comprises the amino acid sequence of SEQ ID NO:71, or

[0367] (ii) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence described in SEQ ID NO:71, or

[0368] (iii) consists of the amino acids described in SEQ ID NO:71; or

[0369] (iv) comprises an amino acid sequence having one or several (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID NO:71, said mutations being, for example, substitutions, deletions or additions, preferably substitutions, for example conservative substitutions, preferably, said mutations are not present in the CDRs of the variable heavy chain region of the anti-CTLA-4 antibody, or preferably not present in the variable heavy chain region;

[0370] and / or

[0371] heavy chain 2

[0372] (i) comprises the amino acid sequence of SEQ ID NO:72 or 74, or

[0373] (ii) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence described in SEQ ID NO:72 or 74, or

[0374] (iii) consists of the amino acids described in SEQ ID NO:72 or 74; or

[0375] (iv) comprising an amino acid sequence having one or several (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID NO: 72 or 74, said mutations being, for example, substitutions, deletions or additions, preferably substitutions, such as conservative substitutions; preferably, said mutations are not present in the CDRs of the anti-TIGIT VHH or in the VHH against TIGIT;

[0376] and / or

[0377] light chain

[0378] (i) comprising the amino acid sequence of SEQ ID NO: 68, or

[0379] (ii) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 68, or

[0380] (iii) consisting of the amino acids of SEQ ID NO: 68; or

[0381] (iv) comprising an amino acid sequence having one or several (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID NO: 68, said mutations being, for example, substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, preferably, said mutations are not present in the CDRs of the light chain variable region of the anti-CTLA-4 antibody or preferably not in the light chain variable region.

[0382] In some embodiments, the bispecific antibody of the invention comprises

[0383] (1) Heavy chain 1, which comprises the amino acid sequence of SEQ ID NO: 71, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 71, or consists of said sequence; and

[0384] (2) Heavy chain 2, which comprises the amino acid sequence of SEQ ID NO: 72 or 74, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 72 or 74, or consists of said sequence; and

[0385] (2) A light chain, which comprises the amino acid sequence of SEQ ID NO: 68, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 68.

[0386] In one embodiment of the present invention, the VHH, heavy chain antibody or bispecific antibody described herein comprises one or more amino acid mutations. In some embodiments, the amino acid mutations include substitution, insertion or deletion of amino acids. Preferably, the amino acid alterations described herein are amino acid substitutions, preferably conservative substitutions.

[0387] In a preferred embodiment, the amino acid mutations of the present invention occur in regions outside the CDRs (e.g., in the FRs). In some embodiments, the amino acid mutations of the present invention occur in the constant region of the antibody heavy chain, such as on the Fc region. In a preferred embodiment, the amino acid mutations on the Fc region enhance the ADCC effect of the antibody.

[0388] In certain embodiments, one or more amino acid mutations can be introduced into the Fc region of the antibodies provided herein to generate Fc region variants to alter one or more functional properties of the antibody, such as serum half-life, complement binding, complement-dependent cytotoxicity, Fc receptor binding, and / or antibody-dependent cytotoxicity. The Fc region variants can include human Fc region sequences (such as human IgG1, IgG2, IgG3 or IgG4 Fc regions) containing amino acid mutations (such as substitutions) at one or more amino acid positions.

[0389] In certain embodiments, it may be necessary to mutate the variable region of the antibody to prevent deamidation, for example, mutating one or two deamidation-prone amino acids (such as aspartic acid) in the variable region, such as in the CDR.

[0390] In certain embodiments, the antibodies provided herein can be further modified to contain other non-protein moieties known and readily available in the art. Suitable moieties for such derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propanediol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, poly(propylene oxide / ethylene oxide) copolymer, polyoxyethylated polyols (such as glycerol), polyvinyl alcohol, and mixtures thereof.

[0391] The VHH antibody, heavy chain antibody or bispecific antibody of the present invention has one or more of the following properties.

[0392] In some embodiments, the anti-TIGIT VHH or heavy chain antibody of the present invention has one or more of the following characteristics

[0393] (i) capable of specifically binding to TIGIT, such as human TIGIT or cynomolgus monkey TIGIT;

[0394] (ii) capable of specifically binding to human TIGIT, such as with high affinity, for example, as measured by the Fortebio detection system, for example, as measured by the method described in Example 4, K D is less than 1 nM, such as less than 0.9 nM or 0.8 nM, and also such as less than 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1 nM, and also such as less than 5×10 -12 M, 4×10 -12 M, 3×10 -12 M, 2×10 -12 M, or 1×10 -12 M;

[0395] (iii) capable of specifically binding to cynomolgus monkey TIGIT, such as with high affinity, for example, as measured by the Fortebio detection system, for example, as measured by the method described in Example 4, K D is less than 6, 5, 4, 3, 2 or 1 nM, and also such as less than 0.9 nM or 0.8 nM, and also such as less than 5×10 -12 M, 4×10 -12 M, 3×10 -12 M, 2×10 -12 M, or 1×10 -12 M;

[0396] (iv) inhibiting the binding of TIGIT (such as human TIGIT) to CD155, such as human CD155, with a blocking activity higher than that of known TIGIT antibodies, such as the Tiragolumab analog of WO2017053748A2;

[0397] (v) binding to cells expressing TIGIT (such as human or cynomolgus monkey TIGIT), with a binding activity higher than that of known TIGIT antibodies, such as the Tiragolumab analog of WO2017053748A2;

[0398] (vi) effectively activating T cells (such as primary T cells, such as CD8+ T cells), such as activating T cells to release cytokines, such as IFNγ;

[0399] (vii) It has a good effect of inhibiting tumors;

[0400] (viii) It has low toxicity.

[0401] In some embodiments, the bispecific antibody of the present invention can specifically bind to TIGIT and PD-1, such as human TIGIT and human PD-1, for example, with high affinity. In some embodiments, the bispecific antibody of the present invention can specifically bind to TIGIT and CTLA-4, such as human CTLA-4 and human TIGIT, for example, with high affinity.

[0402] In some embodiments, the bispecific antibody that specifically binds to TIGIT and PD-1 of the present invention has one or more of the following properties:

[0403] (i) It can specifically bind to TIGIT, such as human and / or cynomolgus monkey TIGIT, for example, with high affinity;

[0404] (ii) It can specifically bind to PD1, such as human PD1, for example, with high affinity;

[0405] (iii) It inhibits the binding of TIGIT (such as human TIGIT) to CD155, such as human CD155, and the blocking activity is higher than that of known TIGIT antibodies, such as the Tiragolumab analog in WO2017053748A2;

[0406] (iv) It blocks the binding of PD1 to PD-L1, such as blocking the binding of human PD1 protein to human PD-L1;

[0407] (v) It binds to cells expressing TIGIT (such as human or cynomolgus monkey TIGIT)

[0408] (vi) It binds to cells expressing TIGIT (such as human or cynomolgus monkey TIGIT), and at the same time binds to cells expressing PD1 (such as human PD1);

[0409] (vii) It has good structural safety, for example, it does not significantly kill cytotoxic T cells (such as CD4+T and CD8+T cells), preferably it cannot significantly induce the killing of CD4+T and CD8+T cells activated by NK cell killing, and the ADCC killing activity is comparable to that of known TIGIT antibodies, such as the Tiragolumab analog in WO2017053748A2;

[0410] (viii) It has good pharmacokinetic characteristics and good drug-forming properties (such as stability);

[0411] (ix) having the activity of a PD1 antibody, such as a known PD1 antibody (e.g., the PD1 antibody disclosed in WO2019219064A); having the activity of the TIGIT VHH or heavy chain antibody of the present invention;

[0412] (x) having a good tumor inhibitory effect;

[0413] (xi) having low toxicity.

[0414] In some embodiments, the bispecific antibody specifically binding to TIGIT and CTLA-4 of the present invention has one or more of the following properties:

[0415] The bispecific antibody specifically binding to TIGIT and CTLA-4 has one or more of the following properties:

[0416] (i) capable of specifically binding to TIGIT, such as human and / or cynomolgus monkey TIGIT, for example with high affinity;

[0417] (ii) capable of specifically binding to CTLA-4, such as human CTLA-4, for example with high affinity;

[0418] (iii) the binding affinity to TIGIT is higher than the binding affinity to CTLA-4 (e.g., one order of magnitude higher)

[0419] (iv) inhibiting the binding of TIGIT (e.g., human TIGIT) to CD155, such as human CD155, with a blocking activity higher than that of a known TIGIT antibody, such as the Tiragolumab analog of WO2017053748A2;

[0420] (v) blocking the binding of CTLA-4 to CD80, such as blocking the binding of human CTLA-4 to human CD80;

[0421] (vi) binding to cells expressing TIGIT (e.g., human or cynomolgus monkey TIGIT)

[0422] (vii) having good structural safety, for example, having no obvious killing effect on cytotoxic T cells (e.g., CD4+ T and CD8+ T cells), preferably not significantly inducing the killing of CD4+ T and CD8+ T cells activated by NK cell killing, and the ADCC killing activity is comparable to that of a known TIGIT antibody, such as the Tiragolumab analog of WO2017053748A2;

[0423] (viii) having good pharmacokinetic characteristics and good drug-likeness (e.g., stability);

[0424] (ix) having the activity of an anti-CTLA-4 antibody, such as a known CTLA-4 antibody (e.g., Ipilimumab);

[0425] (x) having the activity of the TIGIT VHH or heavy chain antibody of the present invention;

[0426] (xi) having a better tumor inhibitory effect;

[0427] (xii) having lower toxicity.

[0428] II. Nucleic Acids Encoding Antibodies and Host Cells Containing the Same

[0429] In one aspect, the present invention provides nucleic acids encoding any of the above VHHs or heavy chain antibodies or bispecific antibodies or any of their chains.

[0430] For example, the nucleic acids of the present invention include nucleic acids encoding amino acid sequences shown in any one of SEQ ID NO: 1, 2, 6, 7, 8, 9, 10, 14-22, 30, 32, 33, 69, 71, 72 or 74, or nucleic acids encoding amino acid sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequences shown in any one of SEQ ID NO: 1, 2, 6, 7, 8, 9, 10, 14-22, 30, 32, 33, 69, 71, 72 or 74.

[0431] As will be appreciated by those skilled in the art, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences. The nucleic acid sequences encoding the molecules of the present invention can be generated by methods well known in the art, such as by de novo solid-phase DNA synthesis or by PCR amplification.

[0432] In one aspect, the present invention provides nucleic acids encoding any of the above single-domain antibodies or VHHs. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid is capable of displaying human TIGTI antigen (and / or cynomolgus monkey) binding ability. In some embodiments, the nucleic acid is operably linked in-frame to a nucleic acid encoding another peptide / polypeptide such that when expressed from a suitable expression vector, a fusion protein or chimeric polypeptide comprising the single-domain antibody or VHH and the other peptide / polypeptide is produced. For example, in some embodiments, the nucleic acid is operably linked in-frame to a nucleic acid encoding an Fc region (e.g., a human Fc region) such that when expressed from a suitable expression vector, a heavy chain antibody comprising the single-domain antibody or VHH and the Fc region is produced.

[0433] For ease of production and purification, the single-domain antibody or VHH may be fused at the N-terminus with a secretory signal peptide and / or a tag peptide facilitating purification, such as a hexahistidine tag or a biotin label or an hFc label.

[0434] In yet another aspect, the present invention provides a nucleic acid encoding any of the above bispecific antibodies. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid is capable of exhibiting the binding ability to human TIGIT and a second antigen (such as human PD-1 or human CTLA-4). In one embodiment, the nucleic acids encoding the heavy and light chains of the bispecific antibody may be in the same vector or in different vectors. In yet another embodiment, the nucleic acids encoding the heavy and light chains of the bispecific antibody may be introduced into the same or different host cells for expression. Thus, in some embodiments, the method for producing the bispecific antibody of the present invention comprises the step of culturing a host cell containing nucleic acids encoding the heavy and light chains under conditions suitable for expressing the heavy and light chains of the molecule to produce the bispecific antibody of the present invention.

[0435] In one embodiment, a vector containing the nucleic acid is provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include but are not limited to viruses, plasmids, cosmids, λ phages, or yeast artificial chromosomes (YACs). In one embodiment, the vector is, for example, a pcDNA vector, such as pcDNA3.1.

[0436] In one embodiment, a host cell containing the nucleic acid or the vector is provided, such as for cloning or expressing a vector encoding a VHH or a heavy-chain antibody or a bispecific antibody. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (such as CHO cells (such as CHO-S) or 293 cells (such as 293F or HEK293 cells)) or other cells suitable for preparing antibodies or fragments thereof. In one embodiment, the host cell is prokaryotic, such as a bacterium, such as Escherichia coli.

[0437] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for the production of antibodies or fragments thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies. For example, fungal and yeast strains in which the glycosylation pathway has been "humanized" result in the production of antibodies with a partially or fully human glycosylation pattern. Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines that have been engineered for suspension growth can be used. Other examples of useful mammalian host cell lines are the simian kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (HEK293, 293F, or 293T cells), etc. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells, CHO-S cells, ExpiCHO, etc.; and myeloma cell lines such as Y0, NS0, and Sp2 / 0. Mammalian host cell lines suitable for producing antibodies are known in the art.

[0438] III. Production and purification of the VHH antibody, heavy-chain antibody, or bispecific antibody of the present invention

[0439] In one embodiment, a method for preparing the VHH antibody, heavy-chain antibody, or bispecific antibody of the present invention is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the VHH or heavy-chain antibody or bispecific antibody (e.g., any one polypeptide chain and / or multiple polypeptide chains) or an expression vector comprising the nucleic acid, as provided above, under conditions suitable for the expression of the VHH antibody, heavy-chain antibody, or bispecific antibody or its chain, and optionally recovering the VHH or heavy-chain antibody or bispecific antibody from the host cell (or host cell culture medium).

[0440] The polynucleotide encoding the polypeptide chain of the VHH antibody, heavy-chain antibody, or bispecific antibody of the present invention can be inserted into one or more vectors for further cloning and / or expression in a host cell. Methods well-known to those skilled in the art can be used to construct the expression vector. Once the expression vector containing one or more nucleic acid molecules of the present invention for expression has been prepared, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, transduction with retroviruses, viral transfection, gene gun, liposome-based transfection, or other conventional techniques.

[0441] The VHH antibodies, heavy-chain antibodies, or bispecific antibodies prepared as described herein can be purified by known prior art methods such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions for purifying a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these are obvious to those skilled in the art.

[0442] The purity of the antibody molecules of the present invention can be determined by any one of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.

[0443] IV. Assay methods for VHH antibodies, heavy-chain antibodies, or bispecific antibodies.

[0444] The VHH, heavy-chain antibodies, or bispecific antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by a variety of assay methods known in the art.

[0445] On the one hand, the target (e.g., antigen) binding activity of the VHH, heavy-chain antibodies, or bispecific antibodies of the present invention is tested, for example, by known methods such as biolayer interferometry, ELISA, etc. Methods known in the art can be used to determine the binding to TIGIT and / or PD1, and exemplary methods are disclosed herein. In some embodiments, radioimmunoassay (RIA), biolayer interferometry (BLI), electrochemiluminescence (ECL), surface plasmon resonance (SPR), or flow cytometry (FACS) is used for measurement.

[0446] The present invention also provides assay methods for identifying the biological activities of VHH, heavy-chain antibodies, or bispecific antibodies. The biological activities are selected from the properties of the VHH, heavy-chain antibodies, or bispecific antibodies of the present invention.

[0447] For example, the binding activity of the antibody molecules of the present invention to cells expressing TIGIT and / or a second antigen (e.g., PD-1 or CTLA-4) can be determined by methods known in the art, such as fluorescence reporters and flow cytometry, or the exemplary methods disclosed in the examples herein. For example, the binding of the antibody molecules of the present invention to TIGIT and / or PD-1 expressed on cells can be determined by the method shown in Example 6.

[0448] For example, for the inhibitory activity of the antibody molecules of the present invention against TIGIT and / or a second antigen (such as PD-1 or CTLA-4), it can be measured by methods known in the art, such as ELISA blocking assays, receptor fluorescence reporter molecule activation assays, cell proliferation assays, or the exemplary methods disclosed in the examples herein. For example, the ELISA blocking assay can be used to measure the blocking activity of the molecule against the binding of human TIGIT and / or human PD-1 to their related receptors, such as the methods shown in Example 5 or 14, and the blocking activity of the molecule against the binding of human TIGIT and / or human CTLA-4 to their related receptors, such as the methods shown in Example 21 or 22.

[0449] For example, for the activation activity of the antibody molecules of the present invention against T cells, it can be measured by methods known in the art, such as T cell activation assay systems, such as primary T cell activation assay systems, for example, by the method shown in Example 7, by detecting the amount of cytokines (such as IFNγ) released by T cells (such as CD8+ T cells).

[0450] For example, for the killing activity or structural safety of the antibody molecules of the present invention against cells, it can be measured by methods known in the art, such as cytotoxicity assay systems, such as NK cell-dependent cytotoxicity assay systems, for example, by the method shown in Example 17.

[0451] The cells used for any of the above in vitro assays are primary cells or cell lines, including cells that naturally express or overexpress TIGIT (such as human or cynomolgus monkey), or CD155, or a second antigen (PD1 or PDL1 (such as human or cynomolgus monkey PD1 or PDL1) or CTLA-4 (such as human CTLA-4)), such as cells that overexpress TIGIT and / or CD155 and / or PD1 and / or PDL1, such as 293 cells, or CHO cells, or Jurkat cells, such as HEK293, or CHO-K1, or Jurkat / NFAT-Luc cells.

[0452] The present invention also provides a method for detecting the drugability of the antibody molecules of the present invention. For example, by detecting the pharmacokinetic characteristics of the antibody molecules, the pharmacokinetic parameters of the antibody molecules in an animal model (such as a rat model) can be obtained by methods known in the art, such as the method shown in Example 18.

[0453] It is understood that any of the above assays can be carried out using a combination of the antibodies of the present invention and other active agents.

[0454] V. Fusion proteins or immunoconjugates, pharmaceutical compositions, drug combinations, and kits of VHH or heavy chain antibodies or bispecific antibodies of the present invention

[0455] In some embodiments, the present invention also provides a fusion protein comprising any VHH or heavy chain antibody or bispecific antibody described herein, for example, it comprises a VHH or heavy chain antibody or bispecific antibody of the present invention, and other molecules (such as other proteins, such as proteins for treatment) linked thereto.

[0456] In some embodiments, the present invention provides an immunoconjugate comprising any VHH or heavy chain antibody or bispecific antibody described herein. Preferably, the immunoconjugate comprises one or more other therapeutic agents (such as cytotoxins or small molecule compounds) or markers.

[0457] In some embodiments, the present invention provides a composition or a drug or a formulation comprising any VHH or heavy chain antibody or bispecific antibody described herein. Preferably, the composition is a pharmaceutical composition.

[0458] In one embodiment, the composition further comprises a pharmaceutically acceptable excipient. In one embodiment, the composition, for example, the pharmaceutical composition, comprises a VHH or heavy chain antibody or bispecific antibody of the present invention, and a combination of one or more other therapeutic agents.

[0459] The composition or drug or formulation of the present invention may also comprise suitable pharmaceutically acceptable excipients, such as pharmaceutically acceptable carriers, pharmaceutical excipients known in the art, including buffers.

[0460] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic agents, absorption delaying agents, etc. that are physiologically compatible.

[0461] For the use and application of pharmaceutically acceptable excipients, see also "Handbook of Pharmaceutical Excipients", 8th Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.

[0462] The composition or drug or formulation of the present invention can be in various forms. These forms include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (such as injections or eye drops), powders or suspensions, liposome formulations and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0463] A drug or formulation comprising a VHH or heavy chain antibody or bispecific antibody described herein can be prepared by mixing a VHH or heavy chain antibody or bispecific antibody of the present invention having the desired purity with one or more optional pharmaceutically acceptable excipients, for example, in the form of a lyophilized preparation or an aqueous solution.

[0464] The composition, medicament or preparation of the present invention may also comprise more than one active ingredient, which is required for the specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents.

[0465] The present invention also provides a pharmaceutical combination or pharmaceutical combination product, which comprises the VHH antibody, heavy chain antibody or bispecific antibody of the present invention, and one or more other therapeutic agents.

[0466] The present invention also provides a kit of medicines comprising the said pharmaceutical combination. For example, the said kit of medicines comprises in the same package:

[0467] - a first container containing a pharmaceutical composition comprising the VHH antibody, heavy chain antibody or bispecific antibody of the present invention;

[0468] - a second container containing a pharmaceutical composition comprising other therapeutic agents.

[0469] VI. Uses of the VHH or heavy chain antibody or bispecific antibody and methods of applying the same.

[0470] The present invention provides, on the one hand, a method for preventing or treating a disease in a subject, comprising administering to the subject the anti-TIGIT VHH, heavy chain antibody or bispecific antibody of the present invention, or an immunoconjugate, composition, medicament or preparation comprising the same.

[0471] In some embodiments, the disease is, for example, a tumor, such as cancer. The cancer can be in the early, middle or late stage or be metastatic cancer. In some embodiments, the cancer can be a solid tumor or a hematological tumor. In some embodiments, the tumor is a tumor or cancer that is tolerant to known drugs, such as known anti-PD-1 antibodies or anti-CTLA-4 antibodies, for example, a refractory tumor or cancer.

[0472] In some embodiments, the patient has PD1 or PDL1 or PDL2 or CTLA-4, and / or TIGIT (for example, at elevated levels, such as nucleic acid or protein levels), (for example, compared with the same tissue of a healthy individual, or compared with the healthy tissue adjacent to the patient).

[0473] In some embodiments, the treatment of the disease will benefit from inhibiting PD1 or PDL1 or PDL2 or CTLA-4, and / or TIGIT at the nucleic acid or protein level.

[0474] In some embodiments, the cancer is a cancer characterized by elevated protein and / or nucleic acid levels (e.g., elevated expression) of PD-1, PD-L1, or PD-L2, or CTLA-4, and / or elevated protein and / or nucleic acid levels (e.g., elevated expression) of TIGIT. For example, the tumor cells of the cancer have elevated protein and / or nucleic acid levels (e.g., elevated expression) of PD-1, PD-L1, and / or PD-L2, or CTLA-4, and / or elevated protein and / or nucleic acid levels (e.g., elevated expression) of TIGIT (e.g., compared to the same tissue of a healthy individual or compared to healthy tissue adjacent to the patient).

[0475] The anti-TIGIT VHH or heavy chain antibody or bispecific antibody of the present invention (and immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) can be administered by any suitable method, including parenteral administration, intranasal administration, and, if local treatment is required, intralesional administration. Parenteral injection or infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous injection or infusion. Depending in part on whether the treatment is short-term or long-term, administration can be by any suitable route, such as by injection, e.g., intravenous or subcutaneous injection. Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

[0476] For the prevention or treatment of a disease, a suitable dose of the anti-TIGIT VHH or heavy chain antibody or bispecific antibody of the present invention (and immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether it is administered for prophylactic or therapeutic purposes, previous treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to a patient in a single treatment or over a series of treatments. In other aspects, the present invention provides the use of the anti-TIGIT VHH or heavy chain antibody or bispecific antibody of the present invention, or an immunoconjugate or composition comprising the same, in the manufacture or preparation of a medicament for use as described herein, e.g., for the prevention or treatment of a related disease or disorder mentioned herein.

[0477] In some embodiments, the anti-TIGIT VHH or heavy chain antibody or bispecific antibody (and immunoconjugates, compositions, pharmaceutical compositions, formulations, etc. comprising the same) can also be administered in combination with one or more other therapies, such as treatment modalities and / or other therapeutic agents, for use as described herein, e.g., for the prevention and / or treatment of a related disease or disorder mentioned herein.

[0478] VII. Diagnosis and Detection

[0479] In one aspect, the present invention also relates to methods for diagnosis and detection of VHH or heavy chain antibodies or bispecific antibodies and compositions for diagnosis and detection comprising the same.

[0480] In certain embodiments, the anti-TIGIT VHH or heavy chain antibodies provided herein can be used to detect the presence of TIGIT in a biological sample. In certain embodiments, the bispecific antibodies provided herein can be used to detect the presence of TIGIT and / or PD1 in a biological sample. In certain embodiments, the bispecific antibodies provided herein can be used to detect the presence of TIGIT and / or CTLA-4 in a biological sample.

[0481] As used herein, the term "detection" includes quantitative or qualitative detection, and exemplary detection methods can involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads conjugated with antibody molecules, ELISA assays, PCR techniques (e.g., RT-PCR). In certain embodiments, the biological sample is a body fluid, such as blood, serum or plasma.

[0482] In certain embodiments, the method comprises contacting a biological sample with a VHH or heavy chain antibody as described herein under conditions that permit binding thereof to TIGIT, and detecting whether a complex is formed between the VHH or heavy chain antibody and TIGIT. The formation of a complex indicates the presence of TIGIT. The method can be an in vitro or in vivo method. In one embodiment, the antibodies of the present invention are used to select a subject suitable for treatment with the VHH or heavy chain antibodies of the present invention, for example, wherein TIGIT is a biomarker for selecting the subject.

[0483] In certain embodiments, the method comprises contacting a biological sample with a bispecific antibody as described herein under conditions that permit binding thereof to TIGIT and / or PD-1, and detecting whether a complex is formed between the antibody and TIGIT and / or PD-1. The formation of a complex indicates the presence of TIGIT and / or PD-1. The method can be an in vitro or in vivo method. In one embodiment, the antibodies of the present invention are used to select a subject suitable for treatment with the bispecific antibodies of the present invention, for example, wherein TIGIT and / or PD-1 is a biomarker for selecting the subject.

[0484] In certain embodiments, the method comprises contacting a biological sample with a bispecific antibody as described herein under conditions that permit binding to TIGIT and / or CTLA-4, and detecting whether a complex forms between the antibody and TIGIT and / or CTLA-4. Formation of the complex indicates the presence of TIGIT and / or CTLA-4. The method can be an in vitro or in vivo method. In one embodiment, the antibodies of the invention are used to select a subject suitable for treatment with the bispecific antibodies of the invention, e.g., wherein TIGIT and / or CTLA-4 is a biomarker for selection of the subject.

[0485] In certain embodiments, a labeled VHH or heavy chain antibody or bispecific antibody is provided. Labels include, but are not limited to, labels or moieties that are directly detectable (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and moieties that are indirectly detectable, such as enzymes or ligands, e.g., by an enzymatic reaction or molecular interaction. In some embodiments, the label is a label such as biotin or hFc.

[0486] In some embodiments provided herein, the sample is obtained prior to treatment with a VHH or heavy chain antibody or bispecific antibody of the invention. In some embodiments, the sample is obtained prior to other therapies. In some embodiments, the sample is obtained during or after treatment with other therapies.

[0487] In some embodiments, TIGIT and / or PD1 is detected prior to treatment, e.g., prior to initiation of treatment or prior to a particular treatment after a treatment interval. In some embodiments, TIGIT and / or CTLA-4 is detected prior to treatment, e.g., prior to initiation of treatment or prior to a particular treatment after a treatment interval.

[0488] In some embodiments, a method of treating a disease of the invention is provided, the method comprising: assaying a subject (e.g., a sample) (e.g., a subject sample) for the presence of TIGIT, thereby determining a TIGIT value, comparing the TIGIT value to a control value (e.g., a value in a normal individual), and if the TIGIT value is greater than the control value, administering to the subject a therapeutically effective amount of a VHH antibody or heavy chain antibody of the invention, optionally in combination with one or more other therapies, thereby treating the disease.

[0489] In some embodiments, a method of treating a disease of the present invention is provided, the method comprising: assaying a subject (e.g., a sample) (e.g., a subject sample) for the presence of TIGIT and / or PD1, thereby determining a TIGIT and / or PD1 value, comparing the TIGIT and / or PD1 value with a control value (e.g., a value in a normal individual), and if the TIGIT and / or PD1 value is greater than the control value, administering to the subject a therapeutically effective amount of the bispecific antibody of the present invention, optionally in combination with one or more other therapies, thereby treating the disease.

[0490] In some embodiments, a method of treating a disease of the present invention is provided, the method comprising: assaying a subject (e.g., a sample) (e.g., a subject sample) for the presence of TIGIT and / or CTLA-4, thereby determining a TIGIT and / or PD1 value, comparing the TIGIT and / or CTLA-4 value with a control value (e.g., a value in a normal individual), and if the TIGIT and / or CTLA-4 value is greater than the control value, administering to the subject a therapeutically effective amount of the bispecific antibody of the present invention, optionally in combination with one or more other therapies, thereby treating the disease.

[0491] VIII. Invention Definition

[0492] It should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0493] To explain this specification, the following definitions will be used, and where appropriate, terms used in the singular may also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0494] The term "about", when used in conjunction with a numerical value, means a numerical value that encompasses a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0495] As used herein, the term "and / or" means any one of the alternatives, or two or more or all of the alternatives.

[0496] As used herein, the term "comprising" or "including" means including the recited element, integer, or step, but not excluding any other element, integer, or step. In this text, when the term "comprising" or "including" is used, unless otherwise specified, the case consisting of the recited element, integer, or step is also covered. For example, when referring to an antibody variable region "comprising" a specific sequence, an antibody variable region consisting of that specific sequence is also intended to be covered.

[0497] As used herein, the term "TIGIT" or "T cell immunoreceptor with Ig and ITIM domains" refers to any native TIGIT from any vertebrate source, including mammals such as primates (e.g., human or cynomolgus monkey) and rodents (e.g., mouse and rat), unless otherwise specified. TIGIT is also known in the art as protein 9 containing V-set and immunoglobulin domains, protein 3 containing V-set and transmembrane domains, VSIG9, VSTM3, and WUCAM. The term covers "full-length", unprocessed TIGIT and any form of TIGIT resulting from processing in the cell. The term also covers naturally occurring variants of TIGIT, such as splice variants or allelic variants. An exemplary amino acid sequence of human TIGIT can be found at UniProt accession number Q495A1. In some embodiments of the invention, human TIGIT comprises the amino acid sequence shown in SEQ ID NO:46, or consists of said sequence. In some embodiments of the invention, cynomolgus monkey TIGIT comprises the amino acid sequence shown in SEQ ID NO:48, or consists of said sequence.

[0498] The term "PD-1" refers to programmed cell death protein 1. The term "PD-1" includes variants, isotypes, homologs, orthologs, and paralogs. For example, in some cases, an antibody specific for the human PD-1 protein may cross-react with the PD-1 protein from a species other than human (e.g., cynomolgus monkey). In other embodiments, an antibody specific for the human PD-1 protein may be fully specific for the human PD-1 protein and not exhibit cross-reactivity with other species or other types, or may cross-react with the PD-1 from some other species but not all other species.

[0499] The term "human PD-1" refers to the human PD-1 sequence, such as the complete amino acid sequence of human PD-1 having Genbank accession number.NP_005009.2.

[0500] The term "cytotoxic T lymphocyte associated protein 4" or "CTLA-4" is an inhibitory receptor upregulated on T cells (Alegre et al., 2001, Nat Rev Immunol 1:220-8). CTLA-4 inhibits the immune response in several ways: it competes with the T cell co-stimulatory receptor CD28 for its ligands CD80 and CD86, and thereby blocks co-stimulation; it sends negative signals to inhibit T cell activation; and it can also capture CD80 and CD86 from opposing cells by trans-endocytosis, resulting in reduced T cell co-stimulation through CD28. It is constitutively expressed on Treg cells and only expressed on conventional T cells after activation. CTLA4 is a key negative regulator of T cells, sharing the same B7 ligands (CD80, CD86) as CD28, but CTLA4 has a higher affinity for B7 molecules. Therefore, CTLA4 competitively binds to B7 molecules on APC cells with CD28 on T cells, thus inhibiting T cell activation (Shunsuke Chikuma. (2017). "CTLA-4, an Essential Immune-Checkpoint for T-Cell Activation" Curr Top Microbiol Immunol 410:99-126.). CTLA4 can also mediate the trans-endocytosis of B7 molecules on the surface of APC cells by Treg cells, thereby reducing the expression of B7 molecules on the surface of APC cells to decrease the activation of CD28 on T cells. In the tumor microenvironment, inhibiting CTLA4 can restore anti-tumor immune responses through two independent but complementary mechanisms. The first is to promote the proliferation and activation of tumor-infiltrating T cells, and the second is to weaken the function of immunosuppressive Treg cells. Ipilimumab can block cytotoxic T lymphocyte associated antigen 4 (CTLA4), but there are many treatment-related adverse events clinically, especially dose-limiting toxicity at higher doses that prevent its maximum anti-tumor activity potential. Current research reports suggest that the possible mechanisms for ipilimumab to produce irAE include: blocking CTLA4 activates T cell clones reactive to self-antigens, leading to symptoms similar to autoimmune diseases; the Fc effect of ipilimumab causes the depletion of tissue-resident Treg cells, thus hindering peripheral tolerance and making patients more prone to irAE after CTLA4 antibody treatment; CTLA4 antibody treatment leads to the endocytosis and degradation of CTLA4 receptors, and ipilimumab significantly downregulates the CTLA4 receptor on the cell surface.

[0501] "Single domain antibody" (sdAb) is used herein to refer to an antibody polypeptide that recognizes and binds to an antigen of interest through a single variable antibody domain, such as a single VH or a single VL. The single variable antibody domain of a single domain antibody can recognize and bind to the antigen of interest without pairing with another antibody variable domain. In this article, a single domain antibody containing a heavy chain variable domain is also called VHH.

[0502] The terms "VHH" or "VHH antibody" are used interchangeably herein and generally refer to an antibody that contains or consists of only one heavy chain variable region and has the activity of binding to an antigen. VHH usually contains three CDRs in four highly conserved framework regions and generally has the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4; CDR1 to CDR3 refer to complementary determining regions 1-3. The CDR sequences in the VHH variable region can be determined according to any CDR definition scheme described in the "Definition" section, and preferably, the boundaries of the three CDRs in the variable region sequence can be defined by IMGT. VHH generally includes only the heavy chain variable domain derived from a heavy chain antibody lacking a light chain, also called a nanobody. The VHH used in the present invention is preferably from a camelid, such as an alpaca, or a humanized form or sequence-optimized form thereof (e.g., an affinity-matured form to increase binding affinity). In some embodiments, the VHH of the present invention is a monovalent monospecific polypeptide molecule consisting of or substantially consisting of a single heavy chain variable region (e.g., the heavy chain variable region of a heavy chain antibody).

[0503] The single-domain antibody or VHH of the present invention can also be included in a larger polypeptide / protein. Examples of polypeptides / proteins containing the VHH of the present invention include, but are not limited to, heavy-chain antibodies (HcAb), bispecific antibodies, or fusion proteins. The "heavy-chain antibody" as described in the present invention refers to an antibody without a light chain. For example, it can include VH-Fc or VH-CH2-CH3 or VH-hinge region-CH2-CH3 from the N-terminus to the C-terminus, or can include VH-CH1-CH2-CH3; it can cover homodimers, such as heavy-chain dimer antibodies without light chains. The VH in the heavy-chain antibody of the present invention can be from a standard antibody or from a single-domain antibody. For example, the VH in the heavy-chain antibody of the present invention can be VHH. In some embodiments, the heavy-chain antibody of the present invention can be a heavy-chain antibody having a framework region and / or a heavy-chain constant region derived from a camelid (llama, camel, especially alpaca), its humanized form or its sequence-optimized form (affinity-matured form), or a fragment thereof (such as a fragment containing at least a part of the constant region). The heavy-chain antibody of the present invention also covers antibodies formed by fusing a heavy-chain variable region or VHH with an Fc region (such as a human IgG Fc region, such as a human IgG1 or IgG4 Fc region). When "VHH" is mentioned in the context of a heavy-chain antibody, bispecific antibody, or fusion protein, it should be understood that it is a part of the bispecific antibody and not a separate molecule.

[0504] As used herein, the term "monospecific" refers to a polypeptide / protein molecule having one or more antigen-binding sites, each of which binds to the same epitope of the same antigen.

[0505] As used herein, the term "multispecific" antibody refers to an antibody having at least two antigen-binding sites, each of the at least two antigen-binding sites binding to a different epitope of the same antigen or to a different epitope of a different antigen. A multispecific antibody is an antibody having binding specificity for at least two different antigen epitopes. In one embodiment, provided herein is such a bispecific antibody having binding specificity for a first antigen and a second antigen.

[0506] The term "multispecific binding molecule" refers to a multispecific binding molecule that is at least bispecific, such as a bispecific binding molecule, that is, the molecule contains at least a first target-binding region and a second target-binding region, wherein the first target-binding region binds to one target or antigen and the second target-binding region binds to another antigen or target. Thus, the molecule according to the present invention has specificity for at least two different antigens or targets. The molecule according to the present invention also covers multispecific molecules containing multiple target-binding regions / binding sites, such as trispecific binding molecules. In some embodiments, the bispecific binding molecule of the present invention is a bispecific antibody.

[0507] As used herein, the term "linker" refers to any molecule that enables the direct connection of different parts of a bispecific binding molecule. Examples of linkers that establish covalent linkages between different molecular moieties include peptide linkers and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol. In some embodiments, the linker is a peptide linker (also referred to as a "linking peptide"), which refers to a short amino acid sequence composed of amino acids, such as glycine (G) and / or serine (S) and / or threonine residues (T) used alone or in combination, or from the hinge region of an immunoglobulin, for connecting the amino acid sequence of the first part of a binding molecule to the second part of the binding molecule. For example, a peptide linker can connect the first target binding region of a binding molecule to the second target binding region. For example, a peptide linker can also connect one part of an antibody to another part of the antibody, such as connecting the light chain variable region to the heavy chain variable region. Preferably, the peptide linker has a length sufficient to connect two entities in such a way that they maintain their conformation relative to each other such that the desired activity is not hindered. In one embodiment, the linking peptide has a length of 5 - 50 amino acids, for example, 10, 15, 20, 25, 30 amino acids in length. In one embodiment, the linking peptide comprises the amino acid sequences (GS)n, (GGS)n, (GSGGS)n, (GGGGS)n, (GGGS)n, and (GGGGS)nG, where n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10. Useful linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. In some embodiments, the peptide linker is (GGS)n, where n = 1, 2, 3, or 4, for example, the sequence shown in SEQ ID NO:44.

[0508] In yet another embodiment, the linking peptide is from the hinge region or a portion of the hinge region of an immunoglobulin, including the native hinge region or a portion thereof, or a mutated hinge region or a portion thereof. In one embodiment, the linking peptide is, for example, the hinge region or a portion thereof (such as EPKSC) of an immunoglobulin (such as IgG, such as IgG1, IgG2, IgG3, or IgG4) or a mutated hinge region or a portion thereof, such as EPKSS. Alternatively, computer programs can be used to simulate the three-dimensional structures of proteins and peptides, or phage display methods can be used to rationally design suitable flexible linking peptides.

[0509] As used herein, the term "target-binding region" refers to any portion of a multispecific binding molecule, such as a bispecific binding molecule, that binds a specific target or antigen. The target-binding region can be, for example, an antibody or immunoglobulin itself or an antibody fragment. Such a target-binding region may or may not have a tertiary structure independent of the remainder of the BsAB and may bind or not bind its target as a separate entity. The target-binding region can also be a receptor or ligand, or a domain of a receptor capable of binding a ligand. In the case of a multispecific antibody or bispecific antibody, the "target-binding region" is also referred to as the "antigen-binding region".

[0510] The terms "whole antibody" or "full-length antibody" are used interchangeably herein and refer to an antibody molecule having the structure of a native immunoglobulin molecule. In the case of a conventional four-chain IgG antibody, the full-length antibody comprises two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of a heavy-chain antibody having only heavy chains and lacking light chains, the full-length antibody comprises two heavy chains (H) interconnected by disulfide bonds. For a conventional four-chain IgG antibody, the full-length antibody heavy chain typically consists of a heavy-chain variable region (abbreviated herein as VH) and a heavy-chain constant region, where the heavy-chain constant region comprises at least three domains, CH1, CH2, and CH3. The full-length antibody light chain consists of a light-chain variable region (abbreviated herein as VL) and a light-chain constant region, where the light-chain constant region consists of one domain, CL. Each heavy-chain variable region VH and each light-chain variable region consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody fragment" includes a portion of a whole antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0511] The term "antigen-binding fragment" of an antibody is a molecule distinct from the full-length antibody, which contains a part of the full-length antibody, but can bind to the antigen of the full-length antibody or compete with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment is derived) for binding to the antigen. Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv, single-chain Fv, diabody, single-domain antibody (sdAb), nanobody. For example, Fab fragments can be obtained by papain digestion of full-length antibodies. In addition, pepsin digestion of intact antibodies below the disulfide bonds in the hinge region produces F(ab')2, which is a dimer of Fab’ and is a bivalent antibody fragment. F(ab')2 can be reduced by disrupting the disulfide bonds in the hinge region under neutral conditions, thereby converting the F(ab')2 dimer into Fab' monomers. Fab' monomers are essentially Fab fragments with a hinge region. Fv fragments consist of the VL and VH domains of a single arm of an antibody. The two domains VL and VH of an Fv fragment can be encoded by separate genes, but recombinant methods can also be used to link these two domains with a synthetic linker peptide to produce them as a single protein chain, and in the single protein chain, the VL region and the VH region pair to form a single-chain Fv (scFv).

[0512] The terms "Fab fragment" or "Fab" are used interchangeably herein to refer to an immunoglobulin fragment composed of two polypeptide chains and containing the variable domain of the immunoglobulin heavy chain VH, the constant domain of the heavy chain CH1, the variable domain of the light chain VL, and the constant domain of the light chain CL, wherein one polypeptide chain contains VH and one constant region selected from CH1 and CL from the N-terminus to the C-terminus, and the other polypeptide chain contains VL and the other constant region selected from CL and CH1 from the N-terminus to the C-terminus, and the VH domain and the VL domain pair to form an antigen-binding site. In this context, the Fab polypeptide chain containing the heavy chain constant region CH1 is also referred to as the "Fab heavy chain"; correspondingly, the Fab polypeptide chain containing the light chain constant region CL is also referred to as the "Fab light chain".

[0513] The term "target" refers to the entity to which a binding molecule is directed. The target can be an antigen, or it can be a ligand or a receptor.

[0514] The term "antigen" refers to a molecule that elicits an immune response. Such an immune response may involve antibody production or activation of specific immune cells, or both. One of ordinary skill in the art will understand that any macromolecule, including substantially all proteins or peptides, can be used as an antigen. In addition, an antigen can be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to the portion of an antigen that specifically interacts with an antibody molecule. A "complementary determining region" or "CDR region" or "CDR" is a region in the variable domain of an antibody that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The CDRs located within the variable domain of the antibody heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable domain of the antibody light chain are referred to as LCDR1, LCDR2, and LCDR3. In a given amino acid sequence of a variable light or heavy chain region, the precise amino acid sequence boundaries of each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment schemes, including, for example: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (available on the world wide web at imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0515] The following are the regional ranges of CDRs defined by the Kabat, AbM, Chothia, Contact, and IMGT schemes.

[0516]

[0517] Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" encompasses the CDR sequences determined in any of the above ways. A CDR can also be determined based on having the same Kabat numbered positions as a reference CDR sequence (such as any of the exemplary CDRs of the present invention). Unless otherwise specified, in the present invention, when referring to the residue positions in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbered positions according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0518] In one embodiment, the CDRs in the VHH or heavy chain antibody of the present invention are determined according to IMGT. The VH CDR and VL CDR in the anti-PD1 antibody of the present invention are determined according to Kabat.

[0519] It should be noted that the boundaries of the CDRs of the variable region of the same antibody obtained based on different assignment schemes may vary. That is, the CDR sequences of the same antibody variable region defined under different assignment schemes are different. Therefore, when referring to an antibody defined by a specific CDR sequence of the present invention, the scope of the antibody also encompasses such an antibody, whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries are different from the specific CDR boundaries defined by the present invention due to the application of different schemes (such as different assignment scheme rules or combinations).

[0520] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs (under the same assignment scheme). However, although the CDRs are different between antibodies, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact, and North methods, a minimal overlapping region can be determined, providing a "minimal binding unit" for antigen binding. The minimal binding unit can be a subpart of the CDR. As will be appreciated by those skilled in the art, through the structure and protein folding of the antibody, the residues of the remaining part of the CDR sequence can be determined. Accordingly, the present invention also contemplates variants of any CDR given herein. For example, in a variant of a CDR, the amino acid residues of the minimal binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or Chothia can be replaced with conservative amino acid residues.

[0521] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" or "Fc region" contains two or three constant domains, namely, the CH2 domain, the CH3 domain, and optionally the CH4 domain. For example, in a native antibody, the immunoglobulin Fc domain contains the second and third constant domains (CH2 domain and CH3 domain) from the heavy chain of antibodies of the IgG, IgA, and IgD classes; or contains the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) from the heavy chains of two antibodies of the IgM and IgE classes. Unless otherwise specified herein, the amino acid residue numbering in the Fc region or the heavy chain constant region is numbered according to the EU numbering system (also known as the EU index) as described in Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) (https: / / pubmed.ncbi.nlm.nih.gov / 5257969 / ), see also http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html. In this document, the terms "Fc region", "Fc portion", and "Fc fragment" do not include the variable heavy chain region VH and the variable light chain region VL of the immunoglobulin, as well as the heavy chain constant region CH1 and the light chain constant region CL, but in some cases may include the hinge region at the N-terminus of the heavy chain constant region.

[0522] The term "chimeric antibody" refers to an antibody molecule in which (a) the constant region or a portion thereof is altered, replaced, or exchanged such that the antigen-binding site is linked to a constant region having a different or altered class, effector function, and / or species origin, or to a completely different molecule (e.g., an enzyme, toxin, hormone, growth factor, drug, etc.) that confers new properties to the chimeric antibody; or (b) the variable region or a portion thereof is altered, replaced, or exchanged with a variable region having a different or altered antigen specificity. For example, a camelid heavy-chain antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing an antigen while having reduced antigenicity in humans compared to the original camelid antibody.

[0523] As used herein, a "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (e.g., a llama monoclonal antibody) while having reduced immunogenicity when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen-binding site and replacing the remaining portions of the antibody with their human counterparts (i.e., replacing the constant region and the portions of the variable region that do not participate in binding with the corresponding portions of a human antibody).

[0524] As used herein, the terms "anti-", "bind", or "specifically bind" mean that the binding action is selective for a target or antigen and can be distinguished from unwanted or non-specific interactions. The ability of a binding site to bind to a specific target or antigen can be measured by an enzyme-linked immunosorbent assay (ELISA) or by conventional binding assays known in the art such as by radioimmunoassay (RIA) or by bio-layer interferometry or MSD assay or surface plasmon resonance (SPR).

[0525] The term "effective amount" refers to such an amount or dose of an antibody or fragment or composition or combination of the present invention that, when administered to a patient in a single or multiple doses, produces the desired effect in a patient in need of treatment or prophylaxis.

[0526] A "therapeutically effective amount" refers to an amount that effectively achieves the desired therapeutic result at the required dose and for the required period of time. A therapeutically effective amount is also an amount in which any toxic or harmful effects of the antibody or antibody fragment or composition or combination are less than the therapeutic beneficial effects. Relative to an untreated subject, a "therapeutically effective amount" preferably inhibits or improves a measurable parameter by at least about 40%, even more preferably at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%.

[0527] "Prophylactically effective amount" means an amount that, when administered at the desired dosage for the desired period of time, effectively achieves the desired prophylactic result. Typically, since prophylactic dosages are administered to a subject prior to or at an earlier stage of a disease, a prophylactically effective amount will be less than a therapeutically effective amount.

[0528] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and progeny derived therefrom. A host cell is any type of cell system that can be used to produce the antibody molecules of the invention, including eukaryotic cells such as mammalian cells, insect cells, yeast cells; and prokaryotic cells such as E. coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0529] As used herein, the term "label" refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and facilitates the detection of the reagent to which it is conjugated or fused. A label can itself be detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, can catalyze a chemical alteration of a detectable substrate compound or composition. The term is intended to encompass both direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody and indirect labeling of a probe or antibody by reaction with another directly labeled reagent. In some embodiments, the label is hFc or biotin.

[0530] "Individual" or "subject" are used interchangeably and include mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0531] An "isolated" antibody or molecule is an antibody or molecule that has been separated from the components of its natural environment. In some embodiments, the antibody or molecule is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC).

[0532] The "percent identity" of an amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a specific amino acid sequence shown in this specification, after aligning the candidate sequence with the specific amino acid sequence shown in this specification and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention, which have a substantial degree of identity relative to the antibody molecules and their sequences specifically disclosed herein, such as an identity of at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or higher. The variants may include conservative changes.

[0533] For polypeptide sequences, "conservative changes" include substitutions, deletions or additions to the polypeptide sequence, but do not substantially alter the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in the replacement of one amino acid with a chemically similar amino acid. Tables of conservative substitutions providing functionally similar amino acids are well known in the art. The following lists 8 groups of amino acids that contain conservative substitutions for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C), methionine (M). In some embodiments, the term "conservative sequence change" is used to refer to an amino acid modification that does not significantly affect or alter the antigen-binding characteristics of the antibody molecule or binding protein molecule of the present invention containing the amino acid sequence for the intended antigen. For example, a conservative modified variant retains at least 80%, 85%, 90%, 95%, 98%, 99% or higher, such as 100 - 110% or higher binding affinity for the intended antigen relative to the parental antibody or binding protein.

[0534] The term "pharmaceutical excipient" refers to diluents, adjuvants (such as Freund's adjuvant (complete and incomplete)), excipients, carriers or stabilizers, etc. administered together with the active substance.

[0535] The term "pharmaceutical composition" refers to a composition that exists in a form that allows the biological activity of the active ingredient contained therein to be effective, and does not contain additional ingredients that are unacceptably toxic to the subject to whom the composition is administered.

[0536] The term "drug combination or combination product" refers to non-fixed combination products or fixed combination products, including but not limited to medicine boxes, pharmaceutical compositions. The term "non-fixed combination" means that the active ingredients (e.g., (i) the immunoconjugates of the present invention, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or at the same or different time intervals, sequentially, in separate entities, wherein such administration provides a prophylactically or therapeutically effective level of two or more active agents in the patient. The term "fixed combination" means that two or more active agents are administered to a patient in the form of a single entity. Preferably, the doses and / or time intervals of the two or more active agents are selected such that the combined use of the components can produce an effect greater than that achieved by using any one of the components alone in the treatment of a disease or disorder. Each component can be in the form of a separate preparation, and the forms of the preparations can be the same or different.

[0537] The term "combination therapy" refers to the administration of two or more therapeutic agents or modalities (e.g., radiotherapy or surgery) to treat the diseases described herein. Such administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration includes co-administering the individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). The powder and / or liquid can be reconstituted or diluted to the desired dose prior to administration. In addition, such administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the treatment regimen will provide the beneficial effects of the drug combination in treating the disorders or conditions described herein.

[0538] The term "anti-tumor effect" refers to a biological effect that can be demonstrated by various means, including but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0539] The terms "tumor" and "cancer" are used interchangeably herein and encompass solid tumors and liquid tumors. The terms "cancer" and "cancerous" refer to or describe a physiological disorder in a mammal that is typically characterized by unregulated cell growth.

[0540] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous", and "tumor" are not mutually exclusive when mentioned herein.

[0541] As used herein, "tumor-associated antigen" refers to an antigenic determinant presented on the surface of a target cell, wherein the target cell is a cell in a tumor, such as a cancer cell, a cell of the tumor stroma.

[0542] As used herein, "treatment" refers to the alleviation, interruption, arrest, mitigation, stoppage, reduction, or reversal of the onset of symptoms, complications, or biochemical indicators of a disease, alleviation of symptoms, or prevention or inhibition of further progression of a disease, condition, or disorder.

[0543] As used herein, "prevention" includes the inhibition of the occurrence or development of a disease or disorder or the symptoms of a particular disease or disorder. In some embodiments, a subject with a family history of cancer is a candidate for a preventive regimen. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug prior to the occurrence of the signs or symptoms of cancer, particularly in subjects at risk of cancer.

[0544] The term "vector" as used herein refers to a nucleic acid molecule capable of replicating another nucleic acid linked thereto. The term includes vectors that are self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they have been introduced. The term "expression vector" refers to a vector containing a recombinant polynucleotide that contains expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporated into recombinant polynucleotides.

[0545] "Subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of a tissue or cell sample can be solid tissue, such as from a fresh, frozen, and / or preserved organ or tissue sample, biopsy sample, or aspirate sample; blood or any blood component; body fluids, such as tears, vitreous humor, cerebrospinal fluid, amniotic fluid (amniotic water), peritoneal fluid (ascites), or interstitial fluid; cells from a subject at any time during pregnancy or development. In some embodiments, the tissue sample is tumor tissue. A tissue sample may contain compounds that are not naturally admixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

[0546] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the invention. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from the specification and drawings and from the appended claims. Detailed Description

[0547] Examples

[0548] Example 1: Alpaca immunization

[0549] Select two healthy, strong, good-spirited and medium-sized alpacas A and B (Chengdu Apark Biotechnology Co., Ltd.) for immunization. Before immunization, 10 mL of blood was collected from each alpaca, and the negative serum was collected for use in detecting the immune titer. During immunization, complete Freund's adjuvant was mixed with 0.5 mg of antigen hTIGIT-Fc (ACRO, catalog number TIT-H5254), and after emulsification, it was injected subcutaneously at multiple points. The second immunization was carried out 21 days after the first immunization. 0.25 mg of antigen hTIGIT-Fc (ACRO, catalog number TIT-H5254) was mixed with incomplete Freund's adjuvant, and after emulsification, it was injected subcutaneously at multiple points. A total of 7 immunizations were carried out, with a 3-week interval between each immunization. Among them, the fifth and seventh immunizations were carried out by mixing purified cynomolgus monkey TIGIT-llmaFc protein (SEQ ID NO: 47) with incomplete Freund's adjuvant.

[0550] Starting from the second immunization, 10 mL of peripheral blood was collected 7 days after each immunization to monitor the immune response. After multiple immunizations, alpaca PBMCs available for library construction were obtained.

[0551] Example 2: Construction of alpaca immune library

[0552] Total RNA was extracted from alpaca peripheral blood by the Trizol method. 5 μg of RNA was reverse transcribed using PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, catalog number 6210A) to obtain cDNA. The reverse-transcribed cDNA was subjected to nested PCR amplification. The first-round PCR obtained VH and VHH products. The VHH fragment of about 750 bp was recovered by cutting the agarose gel, and then the VHH fragment of about 500 bp was obtained by the second-round PCR. The VHH fragment was purified using a DNA product purification kit. The VHH fragment was ligated into a phage vector using enzyme digestion and ligation techniques (digested with NheI and NotI). The ligation molar ratio was vector:VHH = 1:3. A total of 10 electroporations were carried out. Immediately after electroporation, 1 mL of 2YT medium (preheated at 37°C) was added to the electroporation cup for recovery. The electroporation product was aspirated and the electroporation cup was washed with 2YT medium. A total of 10 mL of recovery product was obtained. It was recovered at 37°C and 180 rpm for 60 min. 100 μL was taken and serially diluted to 10-3 and 10-4 to determine the number of library transformants, which was spread on a 90 mm plate, and the rest was spread on 10 200 mm plates. The library capacity was measured the next day. After detection, the constructed library capacity was 6×10 7 CFU, and the library insertion rate was 100%.

[0553] Subsequently, library screening was carried out using phage display technology. After 3 rounds of liquid-phase screening, in the first round, the cynoTIGIT protein (ACRO, catalog number TIT-C5223) labeled with 20 μg of biotin (EZ-Link TM Sulfo-NHS-LC-Biotin kit, Thermo, catalog number A39257) was adsorbed, washed 15 times with 0.1% PBST, and finally eluted with 1 mL of 100 mM triethylamine. 500 μL of 1M pH 7.4 Tris-Hcl was taken for neutralization. 750 μL of the eluted phage was used to infect 5 mL of TG1 in the logarithmic phase, allowed to stand at 37 °C for 30 minutes, then centrifuged at 5000 g for 5 minutes, and 1 mL of the bacterial solution was taken to spread on a 2YT (50 mg / mL carbenicillin + 2% glucose) solid plate. Incubated overnight at 37 °C in an inverted position. The next day, all the colonies on the plate were scraped off with 10 mL of 2YT liquid medium, 1 mL of the bacterial solution was added to 100 mL of 2YT (50 μg / mL carbenicillin + 2% glucose) liquid medium, cultured until the logarithmic phase, and the helper phage M13K07 (NEB, catalog number: N0315S) with an MOI ratio of 20:1 was added, infected at 37 °C for 30 minutes, then centrifuged at 5000 rpm for 10 min, the supernatant was discarded completely, and the precipitate was resuspended with an equal volume of 2YT+Car+K (Car: 50 μg / mL, Kan: 50 μg / mL) medium, and incubated overnight at 30 °C, 220 rpm. The overnight culture was centrifuged at 10000 rpm for 20 min at 4 °C, the supernatant was collected, and the precipitate was discarded. The centrifuge tube was replaced, and centrifuged at 10000 rpm for 20 min at 4 °C, and the supernatant was collected.

[0554] Add PEG8000 / NaCl at 1 / 5 of the volume of the supernatant, mix well, and precipitate on ice bath for more than 2 hours. Centrifuge at 10,000 rpm for 20 min at 4°C, discard the supernatant, and centrifuge once more to completely remove the supernatant. Suspend the precipitate with 1 mL of 1×PBS, add PEG8000 / NaCl at 1 / 5 of the volume for secondary precipitation for 1 h. Centrifuge at 12,000 rpm for 10 min at 4°C, discard the supernatant, and centrifuge once more to completely remove the supernatant. Resuspend the precipitate with 1×PBS according to the amount of the precipitate. Take 10 μL of the phage library and dilute it with 2YT in gradient. Take 10 μL from the 10-8 and 10-9 tubes and add them to 90 μL of TG1 bacterial solution, gently mix well. Let it stand at 37°C for 30 min, and then spread them on the carbenicillin-resistant plates and incubate overnight. The next day, count the clones on the plates and calculate the phage library titer. For the second round of screening, adsorb with 10 μg of biotin-labeled huTIGIT protein, wash with 0.1% PBST for 15 times, and finally elute with 1 mL of 100 mM triethylamine. Take 500 μL of 1 M Tris-Hcl at pH 7.4 for neutralization. Thereafter, amplify according to the method of the first round to obtain the phage sub-library required for the third round of screening. For the third round of screening, adsorb with 10 μg of biotin-labeled cynoTIGIT protein, wash with 0.1% PBST for 15 times, and finally elute with 1 mL of 100 mM triethylamine. Take 500 μL of 1 M Tris-Hcl at pH 7.4 for neutralization.

[0555] After the three rounds of screening, dilute the eluted phages and infect the logarithmic-phase TG1, spread them on the 2YT (50 μg / mL carbenicillin + 2% glucose) plates, and incubate at 37°C overnight. The next day, pick the single clones on the plates and transfer them into the deep-well plates containing 400 μL of 2YT (50 μg / mL carbenicillin) liquid medium for culture. Incubate at 37°C and 220 rpm until the logarithmic phase, add 1 mM IPTG, and induce at 30°C overnight at low temperature. The next day, centrifuge the deep-well plates at 500 g for 5 minutes, and take the supernatant for ELISA detection.

[0556] Dilute huTIGIT-his (ACRO, catalog number TIT-H52H3) and cynoTIGIT-his (ACRO, catalog number TIT-C5223) to a concentration of 4 μg / mL with PBS buffer at pH 7.4, add them to a 96-well ELISA plate at a volume of 50 μL / well, and place them at 4 °C overnight. The next day, after discarding the liquid, add 200 μL / well of 1% skim milk blocking solution diluted with PBS, and incubate in an incubator at 37 °C for 1 hour for blocking. After the blocking is completed, discard the blocking solution, wash the plate 2 times with PBST buffer (PBS at pH 7.4 containing 0.005% tween-20), add 50 μL / well of induced supernatant, incubate in an incubator at 37 °C for 1 hour. After the incubation is completed, discard the reaction solution in the ELISA plate, wash the plate 2 times with PBST, add 50 μL / well of diluted HRP-labeled mouse anti-HA tag secondary antibody (Sinobiological, catalog number 100028-MM10), incubate at 7 °C for 1 hour, wash the plate 2 times with PBST, add 50 μL / well of 1 M H2SO4 to terminate the reaction, read the absorbance value at a wavelength of 450 nm with an ELISA reader, and calculate the number of clones binding to huTIGIT and cynoTIGIT. All the obtained positive clones were sequenced and identified, and all antibodies with different sequences were used as candidate objects to obtain the nucleotide sequences of 1G3-VHH, 6F6-VHH, and 5H2-VHH.

[0557] Insert the coding genes of 1G3-VHH, 6F6-VHH, 5H2-VHH, and EPKSS linker peptides into the expression vector pcDNA3.1(+) containing the Fc sequence of the human IgG1 heavy chain constant region (SEQ ID NO: 40) respectively to obtain plasmids expressing anti-TIGIT heavy chain antibodies (1G3, 6F6, 5H2).

[0558] The amino acid sequence of the anti-TIGIT heavy chain antibody 1G3 is as follows (SEQ ID NO: 7), where the linker peptide is shown in bold and underlined, and the constant region Fc is shown in italics.

[0559] The amino acid sequence of the anti-TIGIT heavy chain antibody 6F6 is as follows (SEQ ID NO: 2), where the linker peptide is shown in bold and underlined, and the constant region Fc is shown in italics.

[0560] The amino acid sequence of the anti-TIGIT heavy chain antibody 5H2 is as follows (SEQ ID NO: 10), where the linker peptide is shown in bold and underlined, and the constant region Fc is shown in italics.

[0561] Example 3: Humanization, expression and purification of anti-TIGIT antibody

[0562] By comparing with the IMGT human heavy-chain variable region germline gene database (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi), heavy-chain variable region germline genes with high homology to the single-domain antibodies 1G3, 6F6, and 5H2 screened in Example 2 were respectively selected as templates, and the CDRs of the single-domain antibodies were transplanted into the corresponding human templates to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CR3-FR4. According to the need, key amino acids in the FR region were reverted to the corresponding amino acids of the nanobody (VHH antibody) to ensure the original affinity, thus obtaining the humanized anti-TIGIT VHH antibody. The determination of amino acid residues in the CDR region was determined and annotated by the IMGT numbering system.

[0563] The humanized heavy-chain template of antibody 1G3 is IGHJ4*01, and after humanization, the humanized antibody 1G3-H1 is obtained. The humanized variable region sequence is as follows:

[0564] Table 1 lists the said sequences and lists the corresponding reversion mutation sites.

[0565]

[0566] The humanized heavy-chain template of antibody 6F6 is IGHV3-48*01, and after humanization, the humanized antibody 6F6-H1 is obtained. The humanized variable region sequence is as follows:

[0567] Table 2 lists the said sequences and lists the corresponding reversion mutation sites.

[0568]

[0569] The humanized heavy-chain template of antibody 5H2 is IGHV3-23*01, and after humanization, the humanized antibody 5H2-H1V2 is obtained. The humanized variable region sequence is as follows:

[0570] Table 3 lists the said sequences and lists the corresponding reversion mutation sites.

[0571]

[0572] The nucleic acids encoding 1G3-H1-VH, 6F6-H1-VH, 5H2-H1V2-VH plus the EPKSS linker peptide were respectively inserted into the expression vector pcDNA3.1(+) containing the coding gene of the human IgG1 heavy-chain constant region Fc sequence to obtain plasmids expressing the full-length humanized heavy-chain antibodies against TIGIT (1G3-H1, 6F6-H1, 5H2-H1V2). According to the manufacturer's product manual instructions, ExpiCHO TMExpression system (ThermoFisher, catalog number A29133)

[0573] The plasmids encoding 1G3; 6F6; 5H2; 1G3-H1; 6F6-H1; 5H2-H1V2 obtained in Examples 2 and 3 above were transfected into ExpiCHO-S cells to express TIGIT antibodies. The cells were cultured for 10 - 12 days after transfection. When the cell viability decreased to 60% - 70%, the supernatant was collected. The antibodies expressed in the supernatant were purified using the MabSelect Sure protein A affinity chromatography system (GE healthcare) to obtain the homodimeric heavy chain antibodies of anti-TIGIT heavy chain antibodies. The purified antibodies were concentrated, sterile filtered, and the purity of the antibody protein was detected by SDS-PAGE and size exclusion to be greater than 95%. The results showed that the purity of the antibody met the requirements and could be used for the next experiment.

[0574] The sequence of the control antibody Tiragolumab was from patent WO2017053748A2. It was codon-optimized and gene-synthesized by General Biosystems (Anhui) Co., Ltd. and cloned into the expression vector pcDNA3.1(+). Subsequently, the above expression and purification techniques were applied to obtain this control antibody, which is hereinafter referred to as Tiragolumab analog in this article.

[0575] Example 4: Affinity experiments of anti-human TIGIT heavy chain antibody with human TIGIT protein and cynomolgus monkey TIGIT protein In this experiment, according to the manufacturer's instructions, the binding affinities of the antibodies to human TIGIT-his (ACRO, catalog number TIT-H52H3) and cynomolgus monkey TIGIT protein (ACRO, catalog number TIT-C5223) were detected using the ForteBio Octet RED96e.

[0576] Briefly, the AHC sensor (ForteBio, catalog number 18-5060) was placed into the Running Buffer (1X PBS Hyclone, catalog number SH30256.01, containing 0.02% Tween20, pH 7.0) and pre-equilibrated for 10 min at room temperature. In a 96-well plate, the kinetic experiment was carried out according to the following steps: a) Balance the baseline with Running Buffer for 100 s, b) Add the heavy chain antibody against human TIGIT diluted with Running Buffer at a final concentration of 5 μg / mL and immobilize for 200 s, c) Balance the baseline with Running Buffer for 300 s, d) Add 100 nM human TIGIT protein and cynomolgus monkey TIGIT protein diluted with Running Buffer to each well, bind for 200 s, and dissociate for 600 s. The experimental data was fitted and calculated using the Fortebio Data Analysis software with a 1:1 binding model.

[0577] Table 4 summarizes the binding affinities of the anti-human TIGIT heavy chain antibody of the present invention with human TIGIT protein and cynomolgus monkey TIGIT protein.

[0578] Table 4

[0579]

[0580]

[0581] Table 4 shows that the anti-human TIGIT heavy chain antibody constructed in this application can specifically bind to human TIGIT protein and cynomolgus monkey TIGIT protein and has a high binding activity.

[0582] Example 5: ELISA method for detecting the inhibition of the binding of anti-human TIGIT heavy chain antibody to human TIGIT protein and CD155

[0583] Coat 96-well plates with 0.5 μg / mL of human TIGIT (M22-P141)-Fc protein (SEQ ID NO: 45) at 50 μL / well and incubate overnight at 4°C. Incubate the plates with blocking buffer (PBS solution containing 1% BSA) at 37°C for 1 h for blocking. After blocking, wash the plates three times with PBST solution (PBS solution containing 0.05% Tween 20). Gradient dilute the anti-TIGIT antibody with diluent (starting concentration is 14 nM, 3-fold dilution, 7 concentration points), and mix it with 1.1 μg / mL (concentration before mixing) CD155-mFc (ACRO, catalog number CD5-H5254) at a volume ratio of 1:1, then add it to the plates and incubate at 37°C for 1 h. After incubation, wash the plates with PBST solution. Dilute the secondary antibody (horseradish peroxidase HRP-labeled affinity-purified goat anti-mouse IgG, Fcγ, Jackson Immuno Research, catalog number 115-035-164) with diluent, add it to the plates and incubate at 37°C for 1 h. After incubation, wash again, develop color with TMB for 15 minutes, then terminate with 1 M H2SO4, and then read the absorbance value of OD450nm - OD620nm in an enzyme-linked immunosorbent assay reader. The results are shown in Figure 1 .

[0584] The results show that the anti-human TIGIT heavy chain antibody can block the binding of human TIGIT protein to CD155, and the blocking activity of the humanized TIGIT antibody is stronger than that of the positive control antibody Tiragolumab analog (from patent

[0585] WO2017053748A2, SEQ ID NO: 50, SEQ ID NO: 51).

[0586] Example 6: Binding activity of anti-TIGIT heavy chain antibody to human and monkey TIGIT cells

[0587] (1) The binding ability of the anti-TIGIT heavy chain antibody of the present invention to human TIGIT protein stably expressed on HEK293 cells (Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee, catalog number GNHu 43) was determined by cell binding experiments.

[0588] This experiment was measured by flow cytometry. First, Lipofectamine TM 2000 (Invitrogen, catalog number 11668019) transfection reagent was used. According to the manufacturer's instructions, the nucleotide encoding the full-length human TIGIT protein (amino acid sequence SEQ ID NO: 46) was constructed into a eukaryotic expression vector and then transfected into HEK293 cells. After 48 hours of transfection, 0.3 μg / mL puromycin (Puromycin Dihydrochloride, Gibco, catalog number A1113802) was used to screen for 3 - 5 days to obtain cells with high expression of human TIGIT protein (i.e., HEK293 / human TIGIT cells). The HEK293 / human TIGIT cells were digested, centrifuged, and collected. After resuspending the cells with PBS, they were inoculated into a 96-well plate (Corning, catalog number 3799), with 1×10 5 cells per well. After centrifuging the 96-well plate, the supernatant was discarded. 100 μL of the antibody to be tested diluted in gradients (the 1G3, 6F6, 5H2, 1G3-H1, 6F6-H1 heavy chain antibodies prepared in Example 3, with the highest concentration of 150 nM, diluted 4-fold, a total of 8 concentration points) was added to the HEK293 / human TIGIT cells. After incubating at 4°C for 30 minutes, the cells were centrifuged (1000 rpm, 5 minutes) and the supernatant was discarded. 100 μL of PBS was added to each well to wash the cells twice, and then 100 μl of a 1:200 diluted fluorescent secondary antibody, R-PE-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch, catalog number 109-116-098), was added. After incubating at 4°C for 30 minutes, the cells were washed twice with PBS, and then 100 μL of PBS was added to each well to resuspend the cells. Finally, the fluorescence signal was detected using a Cytoflex (Beckman) flow cytometer. The Tiragolumab analog prepared above was used as a positive control.

[0589] From Figure 2 the results shown, it can be seen that all the tested TIGIT heavy chain antibodies can bind to HEK293 / human TIGIT cells, with an EC50 ranging from 0.6386 nM to 0.7873 nM, and the binding activity is stronger than that of the positive control Tiragolumab (EC50 is 1.605 nM).

[0590] (2) Determine whether the anti-TIGIT heavy chain antibody in the present invention can bind to cynomolgus macaque TIGIT protein stably expressed on the surface of HEK293 cells (Cell Bank of the Chinese Academy of Sciences Committee for Type Culture Collection, catalog number GNHu 43) through cell binding experiments. First, use Lipofectamine TM 2000 (Invitrogen, catalog number 11668019) transfection reagent. According to the manufacturer's instructions, construct the nucleotide encoding cynomolgus macaque TIGIT protein (amino acid sequence SEQ ID NO: 48) into a eukaryotic expression vector, and then transfect it into HEK293 cells. After 48 hours of transfection, screen with 0.3 μg / mL puromycin (Puromycin Dihydrochloride, Gibco, catalog number A1113802) for 3 to 5 days to obtain cells with high expression of cynomolgus macaque TIGIT protein. For flow cytometry, refer to the above text. Add the serially diluted antibody to be tested (the 1G3, 6F6, 5H2, 1G3-H1, 6F6-H1 heavy chain antibodies prepared in Example 3, initial concentration 150 nM, 4-fold dilution, 8 points) to the cells and incubate at 4°C for 30 minutes. Wash the cells twice with PBS and then add the fluorescent secondary antibody R-PE-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, and incubate at 4°C for 30 minutes. Wash the cells twice with PBS and then resuspend the cells. Finally, detect the fluorescence signal with a Cytoflex (Beckman) flow cytometer. Use Tiragolumab analog as a positive control.

[0591] From Figure 3 the results shown, it can be seen that the tested TIGIT heavy chain antibodies can bind to cynomolgus macaque TIGIT protein expressed on HEK293 cells, and the binding activity is stronger than that of the positive control Tiragolumab.

[0592] Example 7: Activation of primary T cell activity by anti-human TIGIT heavy chain antibody

[0593] To detect the activity of anti-TIGIT antibody in activating T cells to release cytokines, we established a primary T cell activation test system, using CHO-K1 / OKT3 / CD155 cells as target cells and CD8+ T cells isolated from human peripheral blood mononuclear cells PBMCs (Shanghai Rubai Biotechnology Co., Ltd.) as effector cells.

[0594] Construction of CHO-K1 / OKT3 / CD155 target cells: Use Lipofectamine TMInvitrogen transfection reagent (Catalog No. 11668019). According to the manufacturer's instructions, the nucleotide encoding human CD155 protein (amino acid sequence SEQ ID NO: 49) was constructed into a eukaryotic expression vector and then transfected into CHO-K1 cells (ATCC, Catalog No. CCL-61). After 48 hours of transfection, the cells were screened with 4 μg / mL puromycin (Puromycin Dihydrochloride, Gibco, Catalog No. A1113802) for 3 to 5 days to obtain a CHO-K1 / CD155 stable cell line that stably expresses CD155 protein. Then, the nucleotide encoding human OKT3 protein (amino acid sequence SEQ ID NO: 53) was constructed into a eukaryotic expression vector and transfected into the established CHO-K1 / CD155 stable cell line using the same method. After 48 hours of transfection, the cells were screened with 500 μg / mL G418 (Gibco, Catalog No. 10131027) for 10 to 14 days to obtain a CHO-K1 / CD155 stable cell line that highly expresses human OKT3 protein (CHO-K1 / OKT3 / CD155 cells).

[0595] Resuscitate PBMC cells. Isolate CD8+ T cells with reference to the instruction manual of the Miltenyi Biotec cell sorting kit (Catalog No. 130-096-495). After resuspending with 1640 complete medium (Gibco, Catalog No. 22400-071) containing 10% FBS (Gibco, Catalog No. 10099-141C), adjust the cell density to 1E6 / mL and seed 100 μL per well (i.e., 100,000 cells per well) into a 96-well plate (Corning, Catalog No. 3599).

[0596] Digest the cultured target cells CHO-K1 / OKT3 / CD155 with trypsin, centrifuge at 1000 rpm for 5 min to collect the cells, discard the supernatant, resuspend with 1640 complete medium (Gibco, Catalog No. 22400-071) containing 10% FBS (Gibco, Catalog No. 10099-141), and adjust the target cell density to 1×10 5 / ml, plated into a 96-well plate (Corning, catalog number 3599) at 50 μL per well (i.e., 5,000 cells per well). Prepare the test antibody 6F6-H1 and the control antibody Tiragolumab analog prepared in Example 3 at 4 times the final detection concentration. The final detection concentrations of the test antibody are 50 nM and 5 nM, and 50 μL per well is added to the 96-well plate, and then incubated in a cell culture incubator for another 72 h. After the incubation is completed, take out the experimental plate, centrifuge at 2,000 rpm for 3 min to precipitate all the cells to the bottom of the plate, carefully aspirate 100 μL of the supernatant into a new 96-well plate, and detect the level of human IFNγ (Cisbio, catalog number 62HIFNGPEH) factor in the supernatant. The results are shown in Figure 4 , indicating that the anti-TIGIT antibody can activate CD8+ T cells to release cytokines.

[0597] Example 8: Optimization of anti-human TIGIT heavy chain sequence and Fc modification

[0598] Using the 6F6-H1 sequence as a template, the VHH sequence was amplified by PCR and ligated to the N-terminus of the IgG1-Fc constant region. At the same time, the following site mutations were made in this Fc region: S239D, A330L, I332E (according to EU numbering). The finally constructed vector was expressed in eukaryotic cells to obtain the antibody 6F6-DLE (the preparation method is as in Example 3), and its amino acid sequence is shown in SEC ID NO:20. Analysis by the online tool Abysis (http: / / www.abysis.org / abysis / ) shows that the asparagine at position D63 in the CDR2 region of the 6F6-H1 sequence is prone to amide site changes. To eliminate the influence of deamidation, the D63S (according to IMGT coding) mutation was carried out. The variable region sequence of 6F6(D63S) was constructed to the N-terminus of the IgG1-Fc constant region according to the above method. This Fc region also contains the mutation sites S239D, A330L, I332E (according to Kabat numbering), and the formed vector was expressed in eukaryotic cells to obtain the antibody 6F6-DS-DLE, and its amino acid sequence is SEC ID NO:22.

[0599] The above optimized antibodies were expressed using the method in Example 3.

[0600] Example 9: Affinity experiments of the optimized and Fc-modified TIGIT heavy chain antibody with human TIGIT protein and cynomolgus monkey TIGIT protein protein

[0601] In this experiment, according to the manufacturer's instructions, the binding affinities of the antibodies to human TIGIT-his (ACRO, catalog number TIT-H52H3) and cynomolgus monkey TIGIT protein (ACRO, catalog number TIT-C5223) were detected using a ForteBio Octet RED96e.

[0602] Briefly, the AHC sensor (ForteBio, catalog number 18-5060) was placed into the Running Buffer (1X PBS Hyclone, catalog number SH30256.01, containing 0.02% Tween 20, pH 7.0) and pre-equilibrated for 10 min at room temperature. In a 96-well plate, the kinetic experiment was performed according to the following steps: a) baseline was equilibrated with the Running Buffer for 180 s, b) the heavy-chain antibodies against human TIGIT (6F6-DS-DLE, 6F6DLE, and 6F6-H1) diluted with the Running Buffer were added, with a final concentration of 5 μg / mL, and immobilized for 200 s, c) the baseline was equilibrated with the Running buffer for 300 s, d) 100 nM human TIGIT protein and cynomolgus monkey TIGIT protein diluted with the Running Buffer were added to each well, and binding was carried out for 200 s and dissociation for 600 s. The experimental data were fitted and calculated using the 1:1 binding model of Fortebio Data Analysis software.

[0603] Table 5 summarizes the binding affinities of the heavy-chain antibodies against human TIGIT after sequence optimization and Fc modification with human TIGIT protein and cynomolgus monkey TIGIT protein.

[0604] Table 5

[0605]

[0606] Table 5 shows that the VHH antibodies against human TIGIT constructed in this application with PTM modification and Fc modification can specifically bind to human TIGIT protein and cynomolgus monkey TIGIT protein, and there is no significant difference in activity compared with that before modification.

[0607] Example 10: ELISA method for the inhibition of the binding of the optimized and Fc-modified TIGIT heavy chain antibody to human TIGIT protein and CD155 Figure 5

[0608] Coat a 96-well plate with 50 μL / well of 0.5 μg / mL human TIGIT (M22-P141)-Fc (SEQ ID NO: 45) and incubate overnight at 4°C. Incubate the plate with the blocking buffer (PBS solution containing 1% BSA) at 37°C for 1 h for blocking. After blocking, wash the plate three times with PBST solution (PBS solution containing 0.05% Tween 20). Dilute the anti-TIGIT antibody with a diluent in a gradient (starting concentration of 14 nM, 3-fold serial dilution), and mix it with 1.1 μg / mL (concentration before mixing) CD155-mFc (ACRO, catalog number CD5-H5254) at a volume ratio of 1:1, then add it to the plate and incubate at 37°C for 1 h. After incubation, wash the plate with PBST solution. Dilute the secondary antibody (horseradish peroxidase HRP-labeled affinity-purified goat anti-mouse IgG, Fcγ, Jackson ImmunoResearch, catalog number 115-035-164) with a diluent, add it to the plate and incubate at 37°C for 1 h. After incubation, wash again, develop color with TMB for 15 minutes, then terminate with 1 M H2SO4, and then read the absorbance value of OD450nm - OD620nm in an enzyme-linked immunosorbent assay reader. The results are shown in Example 11: Construction of anti-PD1 / TIGIT bispecific antibody .

[0609] The results showed that the heavy-chain antibody against human TIGIT after PTM modification and Fc engineering could block the binding of human TIGIT protein to CD155, and there was no significant difference in the blocking ability compared with that before modification.

[0610] Example 12: Binding activity experiment of anti-PD1 / TIGIT bispecific antibody with human TIGIT protein

[0611] The present invention constructs bispecific antibodies with two structures as shown in Figure 6, wherein the anti-TIGIT antibody part of each bispecific antibody is derived from the above-mentioned humanized 6F6-H1 antibody; the anti-PD1 part is derived from the PD1 antibody 1B12 in Patent WO2019219064A, and the constant regions of the bispecific antibodies are in two forms, IgG1 and IgG4. Such bispecific antibodies are also referred to as "anti-PD1 / TIGIT bispecific antibodies" in this article and are sometimes abbreviated as "bispecific antibodies, bispecific antibodies" in the examples.

[0612] Three bispecific antibodies D1, D4, and E4 with the structure shown in Figure 6 are constructed in this application using standard construction methods, wherein

[0613] The heavy chain (D1-H) of bispecific antibody D1, from the N-terminus to the C-terminus, is in the order of the variable region VH of the anti-PD1 antibody heavy chain, the constant region CH1, the VHH of the anti-TIGIT antibody, and the IgG1 Fc constant region, and its amino acid sequence is SEQ ID NO:30; the light chain (D1-L) of bispecific antibody D1, from the N-terminus to the C-terminus, is in the order of the variable region VL of the anti-PD1 light chain antibody and the constant region CL of the light chain, and its amino acid sequence is SEC ID NO:39;

[0614] The heavy chain (D4-H) of bispecific antibody D4, from the N-terminus to the C-terminus, is in the order of the variable region VH of the anti-PD1 antibody heavy chain, the constant region CH1, the VHH of the anti-TIGIT antibody, and the IgG4 Fc constant region, and its amino acid sequence is SEQ ID NO:32; the light chain (D4-L) of bispecific antibody D4, from the N-terminus to the C-terminus, is in the order of the variable region VH of the anti-PD1 antibody and the constant region CL of the light chain, and its amino acid sequence is SEC ID NO:39;

[0615] The heavy chain of bispecific antibody E4, from the N-terminus to the C-terminus, is in the order of the variable region of the anti-PD1 antibody, the constant region CH1, the IgG4 Fc constant region, and the VHH of the anti-TIGIT antibody, and its amino acid sequence is SEC ID NO:33; the light chain of bispecific antibody E4, from the N-terminus to the C-terminus, is in the order of the variable region of the anti-PD1 antibody and the constant region CL of the light chain, and its amino acid sequence is SEC ID NO:39.

[0616] The following methods are used to construct the expression vectors of each bispecific antibody:

[0617] D1-H: Using the PD1 antibody heavy chain coding sequence (SEQ ID NO:56, corresponding to SEQ ID NO:23 in WO2019219064A) in the patent application publication number WO2019219064A as a template, the nucleotide sequences of the variable region of the PD1 heavy chain and the CH1 segment of the constant region are amplified; using 6F6-H1 as a template, the nucleotide sequence of the variable region VHH of the TIGIT antibody is amplified, and the above two sequences are spliced through the linker GGS by overlap PCR and directly ligated to the expression vector containing the human IgG1 constant regions CH2-CH3 to obtain the D1-H molecule by expression.

[0618] D1-L: Using the sequence encoding the PD1 antibody light chain (SEQ ID NO:39, corresponding to SEQ ID NO:39 in WO2019219064A) in WO2019219064A as a template, the nucleotide sequences of the variable region of the PD1 light chain and the CL segment of the constant region are amplified and ligated to the expression vector to obtain the D1-L molecule by expression.

[0619] D4-H uses the PD1 antibody heavy chain coding sequence in WO2019219064A (SEQ ID NO: 55, corresponding to SEQ ID NO: 24 in WO2019219064A) as a template to amplify the nucleotide sequences of the variable region of the PD1 heavy chain and the CH1 segment of the constant region; uses 6F6-H1 as a template to amplify the nucleotide sequence of the variable region VHH of the TIGIT antibody, and uses the overlap PCR technique to splice the above two sequences through the linker peptide GGS; uses the human IgG4 heavy chain as a template to amplify the constant region sequence of the CH2-CH3 segment, and uses the overlap PCR technique to ligate the above two sequences to an expression vector (there is no linker peptide between the two sequences), and expresses to obtain the D4-H molecule.

[0620] D4-L: Using the sequence encoding the PD1 antibody light chain in WO2019219064A (SEQ ID NO: 39, corresponding to SEQ ID NO: 39 in WO2019219064A) as a template, amplify the nucleotide sequences of the variable region of the PD1 light chain and the CL segment of the constant region, and ligate them to an expression vector, and express to obtain the D4-L molecule.

[0621] E4-H: Using the PD1 antibody heavy chain coding sequence in the patent application publication number WO2019219064A (SEQ ID NO: 56, corresponding to SEQ ID NO: 23 in WO2019219064A) as a template, amplify the nucleotide sequences of the variable region of the PD1 heavy chain and the constant region; use 6F6-H1 as a template to amplify the nucleotide sequence of the variable region VHH of the TIGIT antibody, and use the overlap PCR technique to splice the above two sequences and directly ligate them to an expression vector, and express to obtain the E4-H molecule.

[0622] E4-L: Using the sequence encoding the PD1 antibody light chain in WO2019219064A (SEQ ID NO: 39, corresponding to SEQ ID NO: 39 in WO2019219064A) as a template, amplify the nucleotide sequences of the variable region of the PD1 light chain and the CL segment of the constant region, and ligate them to an expression vector, and express to obtain the E4-L molecule.

[0623] Transfect the expression vectors encoding the heavy and light chains of each group above into ExpiCHO-S cells to express bispecific antibodies. Culture the cells for 10 - 12 days after transfection. When the cell viability drops to 60% - 70%, collect the supernatant, and use the MabSelectSure protein A affinity chromatography system (GE healthcare) to purify the antibodies expressed in the supernatant to obtain D1, D4, and E4 bispecific antibodies. Concentrate the purified bispecific antibodies, filter them aseptically, and detect the purity of the antibody protein by SDS-PAGE and size exclusion to be greater than 95%. The results show that the purity of the antibody meets the requirements and can be used for the next experiment.

[0624] Figure 7

[0625] The relative binding activity of the anti-PD1 / TIGIT bispecific antibody to human TIGIT protein was determined by ELISA.

[0626] Human TIGIT-his (ACRO, catalog number TIT-H52H3) was diluted to 0.5 μg / ml with PBS (HyClone, catalog number SH30256.01) and coated on a 96-well plate, 50 μL / well, and incubated overnight at 4°C. Nonspecific binding sites were blocked by incubating at 37°C with PBS containing 1% BSA for 1 hour. After blocking, the plate was washed three times with PBST (PBS containing 0.05% Tween 20). The anti-PD1 / TIGIT bispecific antibody prepared in Example 11 and Tiragolumab analog (control) (starting concentration of 1.5 nM, 3-fold serial dilution, 7 concentration points) were diluted with binding buffer (PBS containing 0.05% Tween 20 and 0.5% BSA) and incubated with the coated protein at 37°C for 1 hour. After incubation, the plate was washed three times with PBST. The peroxidase-labeled goat anti-human Fc secondary antibody (Jackson Immuno Research, 109-035-098) was diluted to 1:25000 with binding buffer, incubated at 37°C for 1 hour, washed again, developed with TMB for 15 minutes, and terminated with 1M H2SO4.

[0627] The absorbance at 450 nm - 620 nm was measured, and the binding curve of the anti-PD1 / TIGIT bispecific antibody to human TIGIT protein is shown in Example 13: Binding activity experiment of anti-PD1 / TIGIT bispecific antibody with human PD1 protein .

[0628] The results showed that all anti-PD1 / TIGIT bispecific antibodies bound to human TIGIT protein, and D1 had comparable activity to the control.

[0629] Figure 8

[0630] The relative binding activity of the anti-PD1 / TIGIT bispecific antibody to human PD1 protein was determined by ELISA.

[0631] Human PD1-his protein (Sino, catalog number 10377-H08H-100) was diluted to 0.2 μg / ml with PBS (HyClone, SH30256.01), coated on a 96-well plate at 50 μl / well, and incubated overnight at 4°C. Nonspecific binding sites were blocked by incubating at 37°C with PBS containing 1% BSA for 1 hour. After blocking, the plate was washed three times with PBST (PBS containing 0.05% Tween 20). The anti-PD1 / TIGIT bispecific antibody prepared in Example 11 and 1B12 PD1 (IgG1mut) (control) (IgG1 mutant of 1B12 PD1 in WO2019219064A, sequences are SEQ ID NO:39, SEQ ID NO:58) were diluted with binding buffer (PBS containing 0.05% Tween 20 and 0.5% BSA) (starting concentration was 1.5 nM, 3-fold serial dilution, 7 concentration points), and incubated with the coated protein at 37°C for 1 hour. After incubation, the plate was washed three times with PBST. The peroxidase-labeled goat anti-human Fc secondary antibody (Jackson Immuno Research, 109-035-098) was diluted to 1:25000 with binding buffer, incubated at 37°C for 1 hour, washed again, developed with TMB for 15 minutes, and then terminated with 1M H2SO4.

[0632] The absorbance at 450 nm - 620 nm was measured, and the binding curve of the anti-PD1 / TIGIT bispecific antibody to human PD1 is shown in Example 14: Inhibition of the binding activity of anti-PD1 / TIGIT bispecific antibody to human TIGIT protein and CD155 protein .

[0633] The results showed that all anti-PD1 / TIGIT bispecific antibodies bound to human PD1 protein, and D1 had comparable activity to the control.

[0634] Figure 9

[0635] Coat a 96-well plate with 50 μL / well of 0.5 μg / mL human TIGIT-human IgG1Fc protein (SEQ ID NO. 45) and incubate overnight at 4°C. Incubate the plate with blocking buffer (PBS solution containing 1% BSA) at 37°C for 1 h for blocking. After blocking, wash the plate three times with PBST solution (PBS solution containing 0.05% Tween 20). Dilute the anti-PD1 / TIGIT bispecific antibody, control Tiragolumab analog, and 1B12PD1 (IgG1mut) (starting concentration of 28 nM, 3-fold serial dilution, 7 concentration points) with diluent and mix them with CD155 (ACRO, catalog number CD5-H5254) respectively, then add them to the plate and incubate at 37°C for 1 h. After incubation, wash the plate with PBST solution. Dilute the secondary antibody (horseradish peroxidase HRP-labeled affinity-purified goat anti-mouse IgG, Fcγ, Jackson Immuno Research, catalog number 115-035-164) with diluent, add it to the plate and incubate at 37°C for 1 h. After incubation, wash again, develop color with TMB for 15 minutes, then terminate with 1 M H2SO4, and then read the absorbance value of OD450nm - OD620nm in an enzyme-linked immunosorbent assay reader. The results are shown in Example 15: Blocking of the interaction between human PD1 and PD-L1 by anti-PD1 / TIGIT bispecific antibody . Use Tiragolumab analog as the positive control and PD1 monoclonal antibody 1B12PD1 (IgG1mut) as the negative control.

[0636] The results show that D1, D4, and E4 can all block the binding of TIGIT protein to CD155 protein, and the blocking ability of D1 and D4 is better than that of the positive control sample.

[0637] Figure 10

[0638] To evaluate the ability of the anti-PD1 / TIGIT bispecific antibody to block the binding of human PD1 protein to PD-L1, the relative inhibitory activity of the anti-PD1 / TIGIT bispecific antibody against human PD1 protein was determined by ELISA.

[0639] Human PD1 (M1-V170)-human IgG1 Fc protein (SEQ ID NO: 52) was diluted to 0.5 μg / ml with PBS (HyClone, SH30256.01) and coated on a 96-well plate at 50 μl / well and incubated overnight at 4°C. Nonspecific binding sites were blocked by incubating at 37°C with PBS containing 1% BSA for 1 hour. After blocking, the plate was washed three times with PBST (PBS containing 0.05% Tween 20). The anti-PD1 / TIGIT bispecific antibody (at a concentration of 22.5 nM, serially diluted 3-fold with 7 concentration points) was diluted with binding buffer (PBS containing 0.05% Tween 20 and 0.5% BSA), and mixed with PD-L1 protein (Sino, catalog number 10084-H05H) diluted to 0.8 μg / ml at a ratio of 1:1, and then incubated with the coated protein at 37°C for 1 hour. Anti-PD1 monoclonal antibody 1B12PD1 (IgG1mut) was used as a positive control, and IgG (SinoBiological catalog number HG1K) was used as a negative control. After incubation, the plate was washed three times with PBST. The peroxidase-labeled goat anti-mouse Fc secondary antibody (Jackson Immuno Research, 115-035-164) was diluted to 1:10,000 with binding buffer, incubated at 37°C for 1 hour, washed again, developed with TMB for 15 minutes, and then terminated with 1M H2SO4.

[0640] The absorbance at 450 nm - 620 nm was measured, and the inhibition curve of the anti-human PD1 / TIGIT bispecific antibody PD1 is shown in Example 16: Binding activity of anti-PD1 / TIGIT bispecific antibody to human TIGIT and human PD1 cells .

[0641] The results showed that all anti-PD1 / TIGIT bispecific antibodies could block the interaction between human PD1 protein and PD-L1, and the inhibitory activity of all antibodies was comparable to that of the control.

[0642] Figure 11

[0643] (1) The ability of the anti-PD1 / TIGIT bispecific antibody to bind to human TIGIT protein stably expressed on HEK293 cells (Cell Bank of the Chinese Academy of Sciences Committee for Type Culture Collection, catalog number GNHu 43) was determined by a cell binding assay. The experimental procedures and methods were as in Example 6(1), using Tiragolumab analog as a positive control and anti-PD1 monoclonal antibody 1B12PD1 (IgG1mut) as a negative control.

[0644] From Figure 12 the results shown, it can be seen that the tested PD1 / TIGIT bispecific antibodies could all bind to HEK293 / human TIGIT cells.

[0645] (2) To determine whether the anti-PD1 / TIGIT bispecific antibody can bind to the human PD1 protein stably expressed on the surface of Jurkat / NFAT-Luc cells (Jurkat cells stably expressing the NFAT-Luc reporter gene element, the construction method can be found in WO2019219064A), a cell binding assay was performed. The experimental procedures and methods were the same as those in Example 6(1), using the PD1 monoclonal antibody 1B12PD1 (IgG1mut) as a positive control and Tiragolumab analog as a negative control.

[0646] From Example 17: ADCC killing activity of anti-PD1 / TIGIT bispecific antibody against activated CD4+T and CD8+T cells the results shown, it can be seen that the tested PD1 / TIGIT bispecific antibody can bind to Jurkat / NFAT-Luc / hPD1 cells.

[0647] activity Figure 13

[0648] To detect whether the anti-PD1 / TIGIT IgG1 subtype bispecific antibody mediates the activation of CD4+T and CD8+T cells after NK cell killing, a primary NK cell-dependent cytotoxicity assay system was established, using activated CD4+T and CD8+T cells as target cells and human peripheral blood mononuclear cells PBMCs as effector cells.

[0649] Preparation of target cells: Prepare a coating suspension containing 1 μg / mL OKT3 (Invitrogen, catalog number 16-0037-85) and 1 μg / mL anti-CD28 (Biolegend, catalog number 302934) with pre-cooled PBS, take 5 mL and add it to a 10 cm cell culture dish, and incubate overnight at 4 °C. The next day, discard the coating solution and wash it once with pre-cooled PBS for later use. Resuscitate PBMC cells, and separately isolate CD4+T (Miltenyi Biotec, catalog number 130-096-533) or CD8+T cells (Miltenyi Biotec, catalog number 130-096-495) according to the instructions of the Miltenyi cell sorting kit. After resuspending CD4+T cells or CD8+T cells with RPMI 1640 complete medium (Gibco, catalog number 22400-071) containing 10% FBS (Gibco, catalog number 10099-141C), adjust the cell density to 5E5 / mL, then add it to the coated 10 cm cell culture dish and place it in the cell culture incubator for 72 hours.

[0650] Resuscitate PBMC cells, adjust the cell density to 1 - 2×10⁶ / mL with RPMI 1640 complete medium containing 10% FBS, add IL2 (Jiangsu Jin Sili Pharmaceutical Co., Ltd.) and activate overnight (final concentration in the system is 100 IU / mL) to obtain the effector cell suspension.

[0651] The next morning, centrifuge and collect the activated CD4⁺T and CD8⁺T target cells separately, centrifuge at 1000 rpm for 5 min, discard the supernatant, and adjust the target cell density to 2×10 5 / mL with MEM-α test buffer (Gibco, cat. no. 41061 - 029) containing 1% FBS (Gibco, cat. no. 10099 - 141), and seed 50 μL per well (i.e., 10,000 cells per well) into a 96-well plate (Corning, cat. no. 3599). Prepare the test antibody D1 and the control Tiragolumab analog at 4× the final detection concentration, with the highest detection concentration of the antibody being 50 nM, and perform a 5-fold serial dilution to obtain 8 concentration points, and add 50 μL to each well.

[0652] Set up controls for maximum killing of target cells (add 2% Triton 100 lysis buffer to target cells), minimum killing (add test buffer to target cells), and natural killing (add only the effector cell suspension to target cells) simultaneously in the experiment.

[0653] The PBMC effector cell suspension contains 200 IU / mL IL2 (Jiangsu Jin Sili Pharmaceutical Co., Ltd.). In each well containing target cells above, finally add 100 μL of the PBMC effector cell suspension containing IL2 to make the final cell density reach 1×10 6 / mL (effector cell E: target cell T = 10:1), then continue to incubate in a cell culture incubator for 24 h. After the incubation, take out the experimental plate, centrifuge at 2000 rpm for 3 min to sediment all cells to the bottom of the plate, carefully aspirate 50 μL of the supernatant into a new 96-well plate, add 50 μL of LDH detection solution (Roche, cat. no. 11644793001) and incubate at room temperature. When the color changes, read the plate on an F50 microplate reader, with the detection wavelength at OD492 nm and the background wavelength at OD650 nm. The results are shown in Example 18: Pharmacokinetics of anti-PD1 / TIGIT bispecific antibody in rats , indicating that D1 cannot specifically induce NK cell killing of activated CD4⁺T and CD8⁺T cells, and has an ADCC killing activity equivalent to that of Tiragolumab analog, suggesting that the structure of D1 has good safety.

[0654] Example 19: Construction of TIGIT / CTLA4 bispecific antibody

[0655] Evaluate the pharmacokinetic characteristics of D1, D4, and E4 in a rat model.

[0656] In this study, D1, D4, and E4 were intravenously injected into rats (Shanghai Biopharmaceuticals Co., Ltd.) at a dose of 10 mg / kg, and blood samples were collected at different time points from 0 to 336 h (0 to 14 days) (before dosing, 10 min, 30 min, 1 h, 4 h, 8 h, 24 h, 48 h, 7 days, 10 days, 14 days after dosing). All samples were processed into plasma and stored frozen at -70 to -86 °C until analysis.

[0657] Human TIGIT-his (ACRO, catalog number TIT-H52H3) protein was diluted to 0.3 μg / mL with PBS (Biosharp, catalog number BL302A), and 50 μL / well was added to the enzyme-linked immunosorbent assay (ELISA) plate (Costar, catalog number 42592) and incubated overnight at 4 °C. Then, it was incubated for 1 h at 37 °C in PBS solution containing 1% bovine serum albumin (Sangon Biotech Co., Ltd., catalog number: A500023-0025g). After blocking, it was washed 3 times with PBST (PBS containing 0.05% Tween-20).

[0658] D1, D4, or E4 was diluted in serum-containing dilution buffer (containing 0.05% Tween-20, 0.5% bovine serum albumin, 2% v / v rat serum), with an initial concentration of 50 ng / mL, and serially diluted 2-fold to 6 concentration points. A total of 7 concentration points of the antibody solution were used as the standard curve.

[0659] Meanwhile, D1, D4, or E4 was diluted in serum-containing dilution buffer to concentrations of 30 ng / mL, 6 ng / mL, and 1.5 ng / mL, respectively, as high, medium, and low quality controls. All rat sera were diluted with blank mixed rat serum and dilution buffer (PBS containing 0.05% Tween-20 and 0.5% bovine serum albumin) to keep the final antibody concentration at 30 to 1.5 ng / mL.

[0660] The standard curve, quality controls, and plasma samples were added to the ELISA plate and incubated at 37 °C for 1 h.

[0661] Then, it was washed 3 times with PBST. The goat anti-human IgG Fc specific antibody (Jackson ImmunoResearch, catalog number 109-035-098) was diluted 40,000-fold or 20,000-fold in dilution buffer, added to the ELISA plate, and incubated at 37 °C for 1 h, and then washed again with PBST. 50 μL / well of TMB (Thermo, catalog number 34029) was added to the ELISA plate. After 13 minutes, the reaction was terminated with 1 M H2SO4, and the absorbance at 450 to 620 nm was measured. The pharmacokinetic parameters were calculated by software and listed in Tables 6, 7, and 8.

[0662] Human PD1-mFc protein (Sino, catalog number 10377-H05H) was diluted to 0.2 μg / mL with PBS (Biosharp, catalog number: BL302A), and 50 μL / well was added to the enzyme-linked immunosorbent assay (ELISA) plate (Costar, catalog number 42592) and incubated overnight at 4 °C. Then it was incubated for 1 hour at 37 °C in PBS solution containing 1% bovine serum albumin (Shanghai Sangon Biotech, catalog number: A500023-0025g). After the blocking was completed, it was washed 3 times with PBST (PBS containing 0.05% Tween-20). D1, D4, and E4 were diluted in serum-containing dilution buffer (containing 0.05% Tween-20, 0.5% bovine serum albumin, 2% v / v rat serum), starting at a concentration of 50 ng / mL, and serially diluted 2-fold for 6 concentration points, and the antibody solutions at a total of 7 concentration points were used as the standard curve.

[0663] Meanwhile, D1, D4, or E4 was diluted in serum-containing dilution buffer to concentrations of 30 ng / mL, 6 ng / mL, and 1.5 ng / mL respectively, as high, medium, and low quality controls. All rat sera were diluted with blank pooled rat serum and dilution buffer (PBS containing 0.05% Tween-20 and 0.5% bovine serum albumin) to keep the final concentration at 30 - 1.5 ng / mL.

[0664] The standard curve, quality controls, and plasma samples were added to the ELISA plate and incubated at 37 °C for 1 h. Then it was washed 3 times with PBST. The donkey anti-human IgG heavy and light chain specific antibody (Jackson ImmunoResearch, catalog number 709-035-149) was diluted 10,000-fold in dilution buffer, added to the ELISA plate and incubated at 37 °C for 1 h, and then washed again with PBST. 50 μL / well of TMB (Thermo, catalog number 34029) was added to the ELISA plate. After 13 minutes, the reaction was terminated with 1 M H2SO4, and the absorbance at 450 - 620 nm was measured. And the pharmacokinetic parameters were calculated by software and listed in Tables 9, 10, and 11.

[0665] Table 6 Summary of D1 TIGIT Pharmacokinetic Characteristics

[0666]

[0667] Table 7 Summary of D4 TIGIT Pharmacokinetic Characteristics

[0668]

[0669]

[0670] Table 8 Summary of E4 TIGIT Pharmacokinetic Characteristics

[0671]

[0672] Summary of Pharmacokinetic Characteristics of 9D1 PD-1

[0673]

[0674] Summary of Pharmacokinetic Characteristics of 10D4 PD-1

[0675]

[0676] Summary of Pharmacokinetic Characteristics of 11E4 PD-1

[0677]

[0678] The above pharmacokinetic experiments show that the antibody of the present invention has the pharmacokinetic characteristics of general antibodies in the rat body, with good stability and drug-likeness, and is suitable for drug development.

[0679] Example 20: Binding affinity of anti-TIGIT / CTLA-4 bispecific antibody

[0680] The present invention constructs TIGIT / CTLA4 bispecific antibodies with three structures as shown in Figure 14. The variable region part of the anti-TIGIT antibody of each bispecific antibody is derived from 6F6-D63S, and the variable region part of the anti-CTLA4 antibody is derived from Ipilimumab. The constant region of the bispecific antibody is in the form of IgG1 (DLE).

[0681] This application constructs three bispecific antibodies THC4, CT1KH, and CT2KH with the structure shown in Figure 14 using standard construction methods. The heavy chain (named HTC) of the bispecific antibody THC4 is in the order of the variable region of the anti-TIGIT heavy chain VHH, the variable region of the anti-CTLA4 heavy chain VH, the CH1 region of the IgG1 constant region, and the Fc constant region of IgG1 from the N-terminus to the C-terminus. The Fc constant region has a mutation of (S239D, A330L, I332E), abbreviated as "DLE" (reference: "Engineered antibody Fc variants with enhanced effector function. Proc Natl Acad Sci USA. 2006 Mar 14;103(11):4005-10."). The amino acid sequence of the heavy chain HTC is SEC ID NO:69. The light chain (named ipilimumabLC) of the bispecific antibody THC4 is in the order of the variable region VL of the anti-CTLA4 antibody light chain and the light chain constant region CL from the N-terminus to the C-terminus, and its amino acid sequence is SEC ID NO:68.

[0682] The heavy chain 1 (named CK) of the bispecific antibody CT1KH, from the N-terminus to the C-terminus, is in the order of the variable region VH of the anti-CTLA4 antibody heavy chain, the IgG1 constant region CH1, and the IgG1 Fc constant region, where the Fc constant region is subjected to point mutations (S239D, A330L, I332E, T366W, S354C) to form an ADCC-enhanced IgG1 "knob" chain (references: "Engineered antibody Fc variants with enhanced effector function. Proc Natl Acad Sci USA. 2006 Mar 14;103(11):4005-10." and Merchant, A.M., et al. (1998). "An efficient route to human bispecific IgG." Nat Biotechnol 16(7):677-681. "Engineered antibody Fc variants with enhanced effector function"), and the amino acid sequence of the heavy chain CK is SEQ ID NO:71; the heavy chain 2 (named TH) of the bispecific antibody CT1KH, from the N-terminus to the C-terminus, is in the order of the variable region VHH of the anti-TIGIT antibody and the constant region IgG1 Fc, where the constant region IgG1 Fc region is subjected to point mutations (S239D, A330L, I332E, Y349C, T366S, L368A, Y407V) to form an ADCC-enhanced IgG1 Fc "hole" chain (references: "Engineered antibody Fc variants with enhanced effector function. Proc Natl Acad Sci USA. 2006 Mar 14;103(11):4005-10." and Merchant, A.M., et al. (1998). "An efficient route to human bispecific IgG." Nat Biotechnol 16(7):677-681.), and the amino acid sequence of the heavy chain TH is shown in detail in SEQ ID NO:72. The light chain of the bispecific antibody CT1KH (named ipilimumab LC) has an amino acid sequence of SEQ ID NO:68.

[0683] The heavy chain 1 of the bispecific antibody CT2KH (named CK), the detailed amino acid sequence is SEQ ID NO:71; the heavy chain 2 (named 2TH), from the N-terminus to the C-terminus, is the variable region VHH of the anti-TIGIT antibody, linker (SEQ ID NO:73), the variable region VHH of the anti-TIGIT antibody, linker (SEQ ID NO:70) and the constant region IgG1Fc, in which the constant region Fc is subjected to point mutations (S239D, A330L, I332E, Y349C, T366S, L368A, Y407V) to form an ADCC-enhanced IgG1Fc "hole" chain (reference "Engineered antibody Fc variants with enhanced effector function. Proc Natl Acad Sci USA. 2006 Mar 14;103(11):4005-10." and Merchant, A.M., et al. (1998). "An efficient route to human bispecific IgG." Nat Biotechnol 16(7):677-681.), and the amino acid sequence of 2TH is SEQ ID NO:74. The light chain of the bispecific antibody CT2KH (named ipilimumab LC) has the amino acid sequence of SEQ ID NO:68.

[0684] The following methods are used to construct the expression vectors of each bispecific antibody:

[0685] HTC: Using the 6F6-DS-DLE plasmid as a template, amplify the 6F6-D63S VHH fragment, and amplify the ipilimumab VH-CH1 using the ipilimumab-HC as a template. The above two sequences are spliced by the linker GGS using the overlap PCR technique and recombined onto the expression vector containing Fc (Fc contains S239D, A330L, I332E point mutations) to form the HTC complete heavy chain expression vector.

[0686] CK: Using the ipilimumab-HC as a template, amplify the ipilimumab VHCH1, amplify the IgG1Fc constant region using the ipilimumab-H IgG1wtRknob (this constant region contains T366W, S354C point mutations to form the knob structure) as a template, and perform "DLE" point mutations (S239D, A330L, I332E) on the Fc constant region. The above fragments are recombined onto the expression vector to form the heavy chain CK expression vector.

[0687] The TH and 2TH expression vectors are obtained by gene synthesis from General Biosystems (Anhui) Co., Ltd.

[0688] Example 21: Inhibition of the binding activity of anti-TIGIT / CTLA-4 bispecific antibody to human TIGIT protein and human CD155 protein

[0689] In this example, the binding affinity (KD) of the anti-TIGIT / / CTLA-4 bispecific antibody to human TIGIT protein and human CTLA-4 protein was determined using a ForteBio Octet RED 96e (Biolayer Interferometry).

[0690] Briefly, AHC (IgG Fc capture) sensor tips (ForteBio, part number 18-5060) were pre-equilibrated in PBST (PBS, 0.02% Tween 20, pH 7.0) at room temperature for 10 minutes. Kinetic experiments were performed in a 96-well plate and carried out as follows: a) baseline was equilibrated in PBST for 180 seconds, b) 5 μg / mL of anti-TIGIT / CTLA-4 bispecific antibodies (THC4, CT2KH, and CT1KH) and control monoclonal antibodies (6F6-H1 and Ipilimumab) were loaded respectively for 200 s and then stopped, c) baseline was equilibrated for 300 seconds, d) associated with His-tagged human TIGIT (SEC ID NO:) or human CTLA-4 (Sino Biological, part number 11159-H08H) at a concentration of 100 nM for 200 seconds respectively, and e) dissociated in PBST for 600 seconds. The data set was fitted with a 1:1 local fitting model using Fortebio Data Analysis software.

[0691] Table 12 summarizes the binding affinities of anti-TIGIT / CTLA-4 bispecific antibodies (THC4, CT2KH, and CT1KH) and control monoclonal antibodies (6F6-H1 and Ipilimumab analog) to human TIGIT protein and human CTLA-4 protein.

[0692] Table 12

[0693]

[0694] The results showed that THC4, CT2KH, and CT1KH could specifically bind to human TIGIT and CTLA-4, and there was no significant change in the binding activities of TIGIT and CTLA4 compared with monoclonal antibodies.

[0695] activity Figure 15

[0696] A high-binding transparent polystyrene 96-well plate was coated with 0.5 μg / mL human TIGIT (M22-P141)-Fc (SEQ ID NO: 45) in phosphate-buffered saline (PBS) at 50 μL / well and incubated overnight at 4°C. Then the plate was washed once on an automatic plate washer with PBST solution (PBS solution containing 0.05% Tween 20). 200 μL of blocking buffer (PBS solution containing 1% BSA) was added to each well and incubated at 37°C for 1 hour. Serial dilutions of antibodies (6F6-H1, THC4, CT2KH, and CT1KH) and negative control antibody (Ipilimumab analog) (starting concentration: 100 nM (concentration before mixing), 3-fold dilution) were prepared with dilution buffer (PBS + 0.5% bovine serum albumin + 0.05% Tween 20) and mixed with 1.1 μg / mL (concentration before mixing) CD155-mFc (Acro Biosystem, catalog number CD5-H5254) at a volume ratio of 1:1. Then the mixed dilution was added to the 96-well plate and incubated at 37°C for 1 hour. The plate was washed twice with washing buffer PBST on an automatic plate washer. Then HRP-conjugated goat anti-mouse Fc antibody (Jackson Immuno Research, catalog number 115-035-164) diluted with dilution buffer was added to each well of the plate at 50 μL / well. After that, the ELISA plate was incubated at 37°C for 1 hour, and then the plate was washed twice with washing buffer PBST on an automatic plate washer. Finally, 50 μL / well of TMB was added to each well, and the reaction was terminated with 1 M H2SO4. The absorbance values were measured at 450 nm - 620 nm, and the results are shown in Example 22: Inhibition of the binding activity of anti-TIGIT / CTLA-4 bispecific antibody to human CTLA-4 protein and human CD80 protein 。

[0697] The results showed that 6F6-H1, THC4, CT2KH, and CT1KH could block the binding of TIGIT to CD155. And there was no significant difference in the blocking activities of THC4, CT2KH, and CT1KH compared with 6F6-H1.

[0698] activity Figure 16

[0699] High-binding transparent polystyrene 96-well plates were coated with 2 μg / mL human CTLA-4-his (Acro Biosystem, catalog number CT4-H5229) in phosphate-buffered saline (PBS) at 50 μL / well and incubated overnight at 4°C. Then the plates were washed once on an automated plate washer with wash buffer PBST (PBS solution containing 0.05% Tween 20). 200 μL of blocking buffer (PBS solution containing 1% BSA) was added to each well and incubated for 1 hour at room temperature. Serial dilutions of antibodies (THC4, CT2KH, and CT1KH, Ipilimumab analog) and negative control antibody (6F6-H1) (starting concentration: 100 nM (concentration before mixing), 3-fold dilution) were prepared with dilution buffer (PBS + 0.5% bovine serum albumin + 0.05% Tween 20) and mixed with 0.02 μg / mL (concentration before mixing) CD80-mFc (Acro Biosystem, catalog number B71-H52A4) at a volume ratio of 1:1. Then the mixed dilution was added to the 96-well plates and incubated for 1 hour at 37°C. The plates were washed twice with wash buffer PBST on an automated plate washer. Then HRP-conjugated goat anti-mouse Fc antibody (Jackson Immunoresearch, Cat#115-035-164) diluted with dilution buffer was added to each well of the plate at 50 μL / well. After that, the 96-well plates were incubated for 1 hour at 37°C, and then the plates were washed twice with wash buffer PBST on an automated plate washer. Finally, 50 μL / well of TMB was added to each well, and the reaction was terminated with 1 M H2SO4. The absorbance was measured at 450 nm - 620 nm, and the results are shown in Example 23: Binding activity of anti-TIGIT / CTLA-4 bispecific antibody to human TIGIT cells 。

[0700] The results showed that Ipilimumab analog, THC4, CT2KH, and CT1KH could block the binding of CTLA-4 and CD80.

[0701] Figure 17

[0702] Cell binding experiments were performed to determine whether the anti-TIGIT / CTLA-4 bispecific antibodies could bind to human TIGIT protein stably expressed on HEK293 cells (Cell Bank of the Chinese Academy of Sciences Committee for Type Culture Collection, catalog number GNHu 43). The experimental procedures and methods were as described in Example 6(1), and TIGIT monoclonal antibody 6F6-H1 was used as a control.

[0703] From ​ the results shown, the tested TIGIT / CTLA-4 bispecific antibodies THC4, CT2KH, and CT1KH could all bind to HEK293 / hTIGIT cells.

[0704] Sequence Information:

[0705]

[0706]

[0707]

[0708]

[0709]

[0710]

[0711]

[0712]

[0713]

Claims

1. A VHH antibody that specifically binds to TIGIT, comprising (i) three complementarity-determining regions (CDRs) contained in VH shown in SEQ ID NO: 9 or 16, preferably, the CDR sequences are defined according to IMGT; (ii) complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, wherein VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 11, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 12, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:

13.

2. The VHH antibody of claim 1, comprising or consisting of a heavy chain variable region, the heavy chain variable region (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence selected from SEQ ID NO: 9 or 16; or (ii) comprises or consists of the amino acid sequence selected from SEQ ID NO: 9 or 16; or (iii) comprises an amino acid sequence having 1 or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence selected from SEQ ID NO: 9 or 16, preferably, the amino acid changes do not occur in the CDR regions.

3. A heavy chain antibody that specifically binds to TIGIT, comprising the VHH antibody according to any one of claims 1-2. Preferably, the antibody constant region or Fc region is from human IgG1, human IgG2, human IgG3, or human IgG4. Optionally, the VHH antibody is linked to the Fc region through a hinge region or a part thereof. Preferably, the amino acid sequence of the hinge region part is EPKSS (SEQ ID NO: 43). Preferably, the Fc region is the Fc region from human IgG1 or IgG4. Preferably, the Fc region (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 40 or 42; or (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 40 or 42; or (iii) comprising an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 40 or 42; optionally, wherein the Fc region comprises a mutation that improves the effector function of the Fc region, such as a mutation that enhances ADCC, preferably, the mutation is a combination of the following mutations: S239D, A330L, and I332E (EU numbering), preferably, it comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 41 and comprises a combination of the following mutations: S239D, A330L, and I332E (EU numbering).

4. The heavy chain antibody of claim 3, wherein (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NO: 10 or 17; or (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 10 or 17; or (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO: 10 or 17, preferably, the amino acid alterations do not occur in the CDR regions.

5. The VHH antibody of any one of claims 1 - 2, or the heavy chain antibody of any one of claims 3 - 4, wherein the antibody is a chimeric antibody or a humanized antibody.

6. A bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds to TIGIT and comprises a VHH antibody of any one of claims 1-2 and 5, or a heavy chain antibody of any one of claims 3-5; preferably, wherein the second antigen-binding region specifically binds to PD-1, PD-L1 or PD-L2 or CTLA-4, preferably, the second antigen-binding region specifically binds to PD-1, which comprises a PD1 antibody from WO2019219064A or an antigen-binding fragment thereof, and the antigen-binding fragment is, for example, a single-chain Fv, Fab, Fab', (Fab)2, single-domain antibody, VHH or heavy chain antibody of the anti-PD-1 antibody; preferably, the second antigen-binding region specifically binds to CTLA-4, which comprises an Ipilimumab antibody or an antigen-binding fragment thereof, and the antigen-binding fragment is, for example, a single-chain Fv, Fab, Fab', (Fab)2, single-domain antibody, VHH or heavy chain antibody of the anti-CTLA-4 antibody; wherein the VHH antibody is linked to the C-terminus of the Fc fragment of the second antigen-binding region, or linked to the N-terminus of the VH fragment of the heavy chain of the second antigen-binding region, or can be inserted between the Fab fragment (heavy chain of the Fab fragment) and the Fc fragment of the second antigen-binding region, that is, linked to the C-terminus of the heavy chain of the Fab fragment and the N-terminus of the Fc fragment, optionally, the first and second antigen-binding regions are linked by a linker, preferably, the linker comprises an amino acid sequence of (GGS)n, where n is an integer of 1, 2, 3, 4, or 5, preferably n = 1.

7. The bispecific antibody of claim 6, wherein the bispecific antibody has the following structure: Heavy chain: from the N-terminus to the C-terminus, the variable heavy chain region VH of the second antigen antibody - the constant heavy chain region CH1 - the constant heavy chain region Fc - the anti-TIGIT VHH; or From the N-terminus to the C-terminus, the variable heavy chain region VH of the second antigen antibody - the constant heavy chain region CH1 - the anti-TIGIT VHH - the constant heavy chain region Fc; or From the N-terminus to the C-terminus, the anti-TIGIT VHH - the variable heavy chain region VH of the second antigen antibody - the constant heavy chain region CH1 - the constant heavy chain region Fc; Light chain: from the N-terminus to the C-terminus, the variable light chain region of the second antigen antibody - the constant light chain region CL; Preferably, the second antigen is selected from PD-1 or CTLA-4.

8. The bispecific antibody of claim 6, wherein the bispecific antibody has the following structure: Heavy chain 1: from the N-terminus to the C-terminus, the variable heavy chain region VH of the second antigen antibody - the constant heavy chain region CH1 - the constant heavy chain region Fc; Heavy chain 2: one or more (e.g., 2) tandem anti-TIGIT VHH - the constant heavy chain region Fc Light chain: from the N-terminus to the C-terminus, the variable light chain region of the second antigen antibody - the constant light chain region CL; Preferably, the second antigen is selected from PD-1 or CTLA-4, such as CTLA-4.

9. The bispecific antibody of claim 8, wherein the anti-TIGIT-VHH is linked to the N-terminus of the heavy chain constant region Fc via a linker peptide, for example, the linker peptide is the hinge region or a portion thereof from human IgG1, 2, 3, or 4, including natural or mutated hinge regions or portions thereof, for example, from the human IgG1 hinge region, for example, the linker peptide is EPKSS (SEQ ID NO: 43).

10. The bispecific antibody of claim 9 or 9, wherein when heavy chain 2 contains multiple tandem anti-TIGIT single domain antibodies VHH, the individual tandem VHHs can be linked by a linker, preferably, the linker contains the (GGGGS)n amino acid sequence, where n is an integer of 1, 2, 3, 4, or 5, preferably n = 1.

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