Antibodies and immunoconjugates comprising the same with tlr7 / 8 agonists and uses thereof

Abstract

Involving TLR7 / 8 agonists, and immunoconjugates comprising the same with antibodies targeting Trop2 and antibodies targeting FRa, therapeutic and diagnostic uses of the immunoconjugates are also provided.

Description

Antibodies and immunoconjugates containing the same and TLR7 / 8 agonists and their uses Technical Field

[0001] The present invention belongs to the fields of tumor therapy and targeted therapy. Specifically, the present invention provides novel anti-TROP2 antibodies, novel FRα antibodies, and novel TLR (e.g., TLR7 / 8) agonists, as well as immunoconjugates (ISACs) comprising the novel antibodies and / or novel TLR agonists, and compositions containing the conjugates. Furthermore, the present invention provides therapeutic and diagnostic uses of the ISACs.

[0002] Background of the Invention

[0003] TROP2, a trophoblast cell surface antigen also known as tumor-associated calcium signaling protein (TACSTD2), is overexpressed in a variety of human epithelial cancers, including breast, lung, gastric, colorectal, pancreatic, prostate, cervical, head and neck, and ovarian cancers (Yezhe Cheng et al., Frontiers in Oncology, 2022 Dec 23;12:951589). Anti-TROP2 antibody-drug conjugates (ADCs) have been investigated in preclinical and clinical studies for the treatment of cancer, but clinical results have been only modest. For example, Trodelvy (an anti-human TROP2 antibody-SN-38 conjugate) has achieved impressive therapeutic efficacy in the treatment of refractory solid tumors, including an objective response rate (ORR) of 33% in patients with drug-resistant triple-negative breast cancer (TNBC). One of Trodelvy's key mechanisms of action is that the SN-38 (payload) is attached via a pH-sensitive linker, which cleaves and specifically releases the SN-38 in the acidic tumor microenvironment. However, because Trodelvy's linker is not stable enough, the maleimide-mediated linker breaks through thiol exchange under physiological conditions, making Trodelvy's half-life in serum relatively short (about 1 day). Therefore, Trodelvy may have a relatively high off-target effect. Another anti-TROP2 antibody-drug conjugate, Ds-1062a (Dato-DXd, AstraZeneca and Daiichi Sankyo), although effective in treating patients with lung cancer and triple-negative breast cancer without gene mutations, still has problems such as uneven distribution of DAR, which affects drug efficacy, and on-target toxicity in normal tissues.

[0004] Folate receptor α (FRα), also known as folate receptor 1 (FOLR1), belongs to the folate receptor family and was discovered as a folate-binding protein. In addition to transporting folic acid, it is also involved in regulating the proliferation and metastasis of tumor cells. Since folic acid is an important nutrient necessary to maintain cell growth and metabolism, this molecule plays a key role in the synthesis of DNA and RNA. In some rapidly proliferating cancer cells (such as breast cancer, ovarian cancer, lung cancer, etc.), due to the higher demand for folic acid, the expression level of folate receptor α is significantly increased, allowing cancer cells to maintain their abnormally high proliferation rate and metabolic activity. There are currently a number of anti-tumor treatment strategies targeting FOLR1, including monoclonal antibodies, bispecific antibodies, ADC drugs, and CAR-T therapy.

[0005] Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that are primarily expressed on dendritic cells, macrophages, monocytes, natural killer cells, and T lymphocytes. TLRs bind to pathogen-associated molecular patterns (PAMPs) from bacteria, fungi, protozoa, and viruses and serve as a first line of defense against invading pathogens. There are 10 known members of the TLR family in humans. They are type I transmembrane proteins characterized by a leucine-rich extracellular domain and a cytoplasmic tail containing the conserved Toll / interleukin (IL)-1 receptor (TIR) ​​domain. Within this family, TLR3, TLR7, TLR8, and TLR9 are located within endosomes.

[0006] TLR7 is mainly expressed on plasmacytoid cells and also on B- cells, which can be activated by binding to specific small molecule ligands (i.e., TLR7 agonists) or its natural ligands (i.e., single-stranded RNA, ssRNA). The altered immune cell responsiveness may be associated with the decline of the innate immune response during chronic viral infection. Agonist-induced TLR7 activation may thus represent a new approach for treating chronic viral infections (DJ Connolly and L.AJ O'Neill, Current Opinion in Pharmacology 2012, 12: 510-518, PA Roethle et al., J. Med. Chem. 2013, 56, 7324-7333).

[0007] Toll-like receptors (TLRs) can also modify the tumor microenvironment and initiate adaptive anti-tumor immunity.

[0008] However, TLR agonists are poorly tolerated due to toxicities associated with widespread immune activation after systemic administration. There is an urgent clinical need to develop effective and safe TLR agonists, especially TLR7 / 8 agonists, as new antiviral and antitumor immunotherapies to provide more treatment options.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention first provides novel TLR agonists, particularly TLR7 / 8 agonists.

[0011] The present invention also provides novel high-affinity TROP2 antibodies, or antigen-binding fragments thereof.

[0012] The present invention also provides novel high-affinity FRα antibodies, or antigen-binding fragments thereof.

[0013] The present invention also provides a novel immunoconjugate (ISAC) comprising a TLR7 / 8 agonist conjugated to a tumor-targeting antibody. The antibody-immunoagonist conjugate of the present invention is well tolerated in vivo upon systemic administration and induces robust activation of intratumoral myeloid cells, leading to tumor clearance and subsequent immune memory.

[0014] In a first aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to TROP2 (e.g., human TROP2). In some embodiments, it comprises:

[0015] (i) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 7, and / or the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 8; or

[0016] (ii) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 12, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 14.

[0017] In some embodiments of the anti-TROP2 antibody or antigen-binding fragment thereof of the present invention, it comprises a first heavy chain complementarity determining region (HCDR1), a second heavy chain complementarity determining region (HCDR2), a third heavy chain complementarity determining region (HCDR3) and a first light chain complementarity determining region (LCDR1), a second light chain complementarity determining region (LCDR2) and a third light chain complementarity determining region (LCDR3), wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, or respectively consist of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0018] In some embodiments, the anti-TROP2 antibodies or antigen-binding fragments thereof of the present invention bind to TROP2 (eg, human TROP2) with sufficient affinity, for example, with an equilibrium dissociation constant (K D ) binds to TROP2, the K D ≤100nM, ≤10nM, ≤1nM, ≤0.55nM, ≤0.5nM, ≤0.4nM or ≤0.35nM, e.g. 10 -8 M or less, for example 10 -7 M to 10 -10 M, for example, between about 0.1 nM and 1 nM, between greater than 0.3 and 0.7 nM, between about 0.4 and 0.6 nM, between about 0.4 and 0.5 nM, between about 0.5 and 0.6 nM, such as detected by ForteBio. In some embodiments, TROP2 is human, mouse, or cynomolgus monkey TROP2. In some embodiments, the antibody binding affinity is determined using biointerferometry.

[0019] In some embodiments, the anti-TROP2 antibodies or antigen-binding fragments thereof of the present invention bind to TROP2 expressed on the surface of a cell.

[0020] In some embodiments, the anti-TROP2 antibodies or antigen-binding fragments thereof of the present invention can induce endocytosis, such as antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention can inhibit and / or reduce the growth and / or volume of tumors in vivo.

[0021] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and / or a light chain variable region VL, wherein VH

[0022] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 7 or 12; or

[0023] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 7 or 12; or

[0024] (iii) comprising or consisting of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 7 or 12, preferably, the amino acid changes do not occur in the CDR regions; and / or

[0025] Among them VL

[0026] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 8 or 14; or

[0027] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 8 or 14; or

[0028] (iii) comprises or consists of 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 changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 8 or 14, preferably, the amino acid changes do not occur in the CDR regions.

[0029] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof of the present invention comprises VH and VL, wherein VH comprises or consists of the amino acid sequence shown in SEQ ID NO:7, and VL comprises or consists of the amino acid sequence shown in SEQ ID NO:8.

[0030] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region and / or a light chain constant region.

[0031] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof of the present invention is an IgG antibody.

[0032] In some embodiments, the heavy chain constant region of the anti-TROP2 antibody or antigen-binding fragment thereof of the present invention is from IgG1 or IgG2 or IgG3 or IgG4, such as IgG1.

[0033] In some embodiments, the anti-TROP2 antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain and a light chain, wherein the heavy chain

[0034] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 9 or 15;

[0035] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 9 or 15; or

[0036] (iii) comprising or consisting of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 9 or 15; and / or

[0037] light chain

[0038] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 10 or 16;

[0039] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 10 or 16; or

[0040] (iii) comprises an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 10 or 16, or consists of said amino acid sequence.

[0041] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO:9, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:10.

[0042] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 15, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 16.

[0043] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof of the present invention is a chimeric antibody or a humanized antibody.

[0044] In some embodiments, the antigen-binding fragment of the anti-TROP2 antibody of the present invention is, for example, Fv, Fab, Fab', Fab'-SH, F(ab')2; dAb (domain antibody); linear antibody; single-chain antibody (e.g., scFv); single-domain antibody such as VHH; bivalent antibody or fragment thereof; or camelid antibody.

[0045] In some embodiments, the present invention also provides a fusion protein comprising the anti-TROP2 antibody or antigen-binding fragment thereof of the present invention.

[0046] In some embodiments, the present invention also provides an immunoconjugate comprising an anti-TROP2 antibody or antigen-binding fragment thereof of the present invention, and further comprising a payload coupled to the antibody or antigen-binding fragment thereof. In some embodiments, the payload is a toxin, a small molecule drug / agent, a cytotoxic agent, an apoptotic agent, a chelating agent, an immunomodulator / immunostimulator / immunoagonist, an oligonucleotide, a polypeptide, a peptide epitope, a radionuclide, a prodrug, etc. In some preferred embodiments, the payload is an immunostimulator / immunoactivator. In some more preferred embodiments, the payload is a TLR agonist. In some more preferred embodiments, the payload is a TLR7 / 8 agonist.

[0047] In some embodiments, the present invention relates to an isolated nucleic acid encoding an anti-TROP2 antibody or antigen-binding fragment thereof of the present invention.

[0048] In some embodiments, the present invention relates to a vector comprising the nucleic acid of the present invention, preferably the vector is an expression vector.

[0049] In some embodiments, the present invention relates to a host cell comprising a nucleic acid or vector of the present invention, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells (e.g., 293 cells or CHO cells, such as CHO-K cells or HEK293 cells) or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

[0050] In some embodiments, the present invention relates to a method for preparing an anti-TROP2 antibody or an antigen-binding fragment thereof, the method comprising

[0051] a) culturing the host cell of the present invention under conditions suitable for expressing a nucleic acid encoding an anti-TROP2 antibody or antigen-binding fragment thereof of the present invention,

[0052] b) optionally isolating said antibody or antigen-binding fragment thereof,

[0053] c) Optionally, the method further comprises recovering the anti-TROP2 antibody or antigen-binding fragment thereof from the host cell. Optionally, the antibody is purified, for example, by Protein A purification.

[0054] In another aspect, the present invention provides an anti-FRα antibody or an antigen-binding fragment thereof.

[0055] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention specifically binds to FRα (e.g., human FRα or cynomolgus monkey FRα). In some embodiments, the binding affinity of the anti-FRα antibody or antigen-binding fragment thereof of the present invention to FRα (e.g., human FRα) is K. D The value is less than or equal to about 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM or 2nM, or greater than about 0.5nM or 1nM, or between the values. In some embodiments, the binding affinity of the antibodies of the present invention is determined by thin-layer biofilm interferometry technology, such as (ForteBio Octet).

[0056] In some embodiments, the anti-FRα antibodies or antigen-binding fragments thereof of the present invention are capable of effectively binding to FRα, such as human FRα.

[0057] In some embodiments, the anti-FRα antibodies or antigen-binding fragments thereof of the present invention comprise three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the anti-FRα antibodies or antigen-binding fragments thereof of the present invention comprise three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, the anti-FRα antibodies or antigen-binding fragments thereof of the present invention comprise three complementarity determining regions (HCDRs) from the heavy chain variable region and three complementarity determining regions (LCDRs) from the light chain variable region.

[0058] In some aspects, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH). In some aspects, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region (VL). In some aspects, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain variable region comprises three complementary determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementary determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0059] In some embodiments, the anti-FRα antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody heavy chain constant region. In some embodiments, the anti-FRα antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody light chain constant region. In some embodiments, the anti-FRα antibodies or antigen-binding fragments thereof of the present invention further comprise a heavy chain constant region and a light chain constant region.

[0060] In some embodiments, the heavy chain variable region of the present invention:

[0061] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 33; or

[0062] (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 33; or

[0063] (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any one of (i) to (ii) or consists of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR region.

[0064] In some embodiments, the light chain variable region of the present invention

[0065] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 34; or

[0066] (ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 34;

[0067] or

[0068] (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from any one of (i) to (ii), or consists of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR region.

[0069] In some embodiments, the three complementarity determining regions (HCDRs) of the present invention, HCDR1, HCDR2, and HCDR3, from the heavy chain variable region are selected from

[0070] (i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 33; or

[0071] (i) a sequence comprising 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 HCDR regions relative to the sequence of any one of (i),

[0072] wherein the HCDRs can be determined according to any scheme for determining CDRs, for example, according to the schemes of Kabat, AbM, Chothia, Contact or IMGT or a combination thereof;

[0073] For example, the HCDR1 is determined according to a union of the Kabat and Chothia schemes, and the HCDR2 and HCDR3 are each determined according to the Kabat scheme.

[0074] In some embodiments, the three complementarity determining regions (LCDRs) of the present invention, LCDR1, LCDR2, and LCDR3, from the light chain variable region are selected from

[0075] (i) three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 34, or

[0076] (i) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three LCDR regions relative to the sequence of any one of (i),

[0077] wherein the LCDR can be determined according to any scheme for determining CDRs, for example, according to the schemes of Kabat, AbM, Chothia, Contact or IMGT, or a combination thereof;

[0078] For example, the LCDR1, LCDR2 and LCDR3 are each determined according to the Kabat protocol.

[0079] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the invention comprises:

[0080] The three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO: 33, and the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO: 34;

[0081] wherein the HCDR and LCDR can be determined according to any scheme for determining CDRs, for example, according to the schemes of Kabat, AbM, Chothia, Contact or IMGT or a combination thereof;

[0082] For example, the HCDR1 is determined according to a union of the Kabat and Chothia schemes, the HCDR2 and HCDR3 are each determined according to the Kabat scheme, and the LCDR1, LCDR2, and LCDR3 are each determined according to the Kabat scheme.

[0083] In some embodiments, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 27, or HCDR1 comprises an amino acid sequence having one, two or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 27.

[0084] In some embodiments, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 28, or HCDR2 comprises an amino acid sequence having one, two or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 28.

[0085] In some embodiments, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 29, or HCDR3 comprises an amino acid sequence having one, two or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 29.

[0086] In some embodiments, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 30, or LCDR1 comprises an amino acid sequence having one, two or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 30.

[0087] In some embodiments, LCDR2 comprises the amino acid sequence of SEQ ID NO: 31, or consists of the amino acid sequence, or LCDR2 comprises an amino acid sequence having one, two or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 31.

[0088] In some embodiments, LCDR3 comprises the amino acid sequence of SEQ ID NO: 32, or consists of the amino acid sequence, or LCDR3 comprises an amino acid sequence having one, two or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 32.

[0089] In some specific embodiments, the anti-FRα antibody of the invention comprises a first heavy chain complementarity determining region (HCDR1), a second heavy chain complementarity determining region (HCDR2), a third heavy chain complementarity determining region (HCDR3), and a first light chain complementarity determining region (LCDR1), a second light chain complementarity determining region (LCDR2), and a third light chain complementarity determining region (LCDR3), wherein

[0090] The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively contain the amino acid sequences shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or respectively consist of the amino acid sequences shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32.

[0091] In some embodiments, the VH of the invention comprises HCDR1, HCDR2, HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein;

[0092] The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively contain the amino acid sequences shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or respectively consist of the amino acid sequences shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32.

[0093] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises:

[0094] A VH comprising the amino acid sequence shown in SEQ ID NO: 33, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consisting of said amino acid sequence, and / or a VL comprising the amino acid sequence shown in SEQ ID NO: 34, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consisting of said amino acid sequence.

[0095] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 33, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 34.

[0096] In one embodiment of the invention, the amino acid changes described herein include amino acid replacements, insertions, or deletions. In some embodiments, the amino acid changes described herein are conservative amino acid changes. Preferably, the amino acid changes described herein are amino acid replacements, preferably conservative replacements. In a preferred embodiment, the amino acid changes described herein occur in regions outside the CDRs (e.g., in the FRs). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region.

[0097] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention further comprises an antibody heavy chain. In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention further comprises an antibody light chain. In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain and a light chain. In some embodiments, the antibody heavy chain of the present invention comprises a heavy chain variable region and a heavy chain constant region, or consists of a heavy chain variable region and a heavy chain constant region. In some embodiments, the antibody light chain of the present invention comprises a light chain variable region and a light chain constant region, or consists of a light chain variable region and a light chain constant region. In some embodiments, the antibody of the present invention comprises two heavy chains and two light chains, or consists of two heavy chains and two light chains. In some embodiments, the antibody of the present invention comprises two identical heavy chains and two identical light chains, or consists of them.

[0098] In some embodiments, the heavy chain constant region of an anti-FRα antibody of the present invention is an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as a human IgG1, IgG2, IgG3, or IgG4 constant region. In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region is an IgG1 heavy chain constant region, such as an amino acid sequence as set forth in SEQ ID NO: 46, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0099] In some embodiments, the anti-FRα antibody light chain constant region of the present invention is a lambda or kappa light chain constant region, preferably a kappa light chain constant region, such as a human lambda or kappa light chain constant region. In some embodiments, the light chain constant region is a (human) lambda light chain constant region. In some embodiments, the lambda light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 38 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 38 and does not comprise a cysteine ​​mutation. In some embodiments, the light chain constant region is a (human) kappa light chain constant region. In some embodiments, the kappa light chain constant region comprises the amino acid sequence set forth in SEQ ID NO:45, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:45.

[0100] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention is an antibody or antigen-binding fragment thereof having a cysteine ​​mutation in the light chain constant region, wherein the antibody or antigen-binding fragment thereof comprises one or two Lambda light chain constant regions and has a cysteine ​​substitution at position 160 (EU numbering) of the Lambda light chain constant region (LLC160C or LLC160), and / or has a cysteine ​​substitution at position 166 (EU numbering) of the Lambda light chain constant region (LLC166C or LLC166).

[0101] In some embodiments, the light chain constant region of the anti-FRα antibody or antigen-binding fragment thereof of the present invention has a cysteine ​​mutation at position 160 (Eu numbering) relative to the wild-type Lambda light chain constant region, such as the amino acid sequence shown in SEQ ID NO: 38.

[0102] In some embodiments, the Lambda light chain constant region with a cysteine ​​mutation at position 160 comprises the amino acid sequence set forth in SEQ ID NO:39, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:39 and comprises the amino acid sequence VKAGVCTTTPS (SEQ ID NO:42).

[0103] In some embodiments, the light chain constant region of the anti-FRα antibody or antigen-binding fragment thereof of the present invention has a cysteine ​​mutation at position 166 (Eu numbering) relative to the wild-type Lambda light chain constant region, such as the amino acid sequence shown in SEQ ID NO: 38.

[0104] In some embodiments, the Lambda light chain constant region with a cysteine ​​mutation at position 166 comprises the amino acid sequence set forth in SEQ ID NO:40, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:40 and comprises the amino acid sequence TTTPSCQSNNK (SEQ ID NO:43).

[0105] In some embodiments, the light chain constant region of the anti-FRα antibody or antigen-binding fragment thereof of the present invention has cysteine ​​mutations at positions 160 and 166 (Eu numbering) relative to the wild-type Lambda light chain constant region, such as the amino acid sequence shown in SEQ ID NO: 38.

[0106] In some embodiments, the Lambda light chain constant region having cysteine ​​mutations at positions 160 and 166 comprises the amino acid sequence set forth in SEQ ID NO:41, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:41 and comprises the amino acid sequence VKAGVCTTTPSCQSNNK (SEQ ID NO:44).

[0107] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise

[0108] (i) one or both Lambda light chain constant regions having a cysteine ​​mutation at position 160 (Eu numbering); and / or

[0109] (ii) one or both Lambda light chain constant regions with a cysteine ​​mutation at position 166 (Eu numbering).

[0110] In some embodiments, the anti-FRα antibodies or antigen-binding fragments thereof of the present invention may also include modifications in the heavy chain constant region or the Fc region contained therein that alter binding affinity for one or more Fc receptors. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In some embodiments, the Fc region comprises mutations that reduce binding to Fcγ receptors. In further preferred embodiments, the Fc fragment may have mutations that result in increased serum half-life, such as mutations that improve binding of the Fc fragment to FcRn.

[0111] The anti-FRα antibody or antigen-binding fragment thereof of the present invention can be any form of antibody or antigen-binding fragment thereof known in the art, such as monoclonal, chimeric, humanized, fully human, bispecific, or multispecific antibody or antibody fragment thereof.

[0112] In some embodiments, the heavy chain of an anti-FRα antibody of the invention comprises

[0113] comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 35;

[0114] comprising or consisting of an amino acid sequence selected from SEQ ID NO: 35; or

[0115] An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 35.

[0116] In some embodiments, the light chain of an anti-FRα antibody of the invention comprises

[0117] comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 36 or 37;

[0118] comprising or consisting of an amino acid sequence selected from SEQ ID NO: 36 or 37; or

[0119] An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 36 or 37.

[0120] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises two identical heavy chains and two identical light chains, wherein

[0121] The heavy chain comprises a VH as described herein, or a HCDR1, HCDR2, and HCDR3 as described herein, and an Fc region and CH1 as described herein, or a heavy chain constant region as described herein; and / or

[0122] The light chain comprises a VL described herein or LCDR1, LCDR2, and LCDR3 described herein, and a kappa light chain constant region described herein.

[0123] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises two identical heavy chains and two identical light chains, wherein

[0124] The heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 35, and / or

[0125] The light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:36.

[0126] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises two identical heavy chains and two identical light chains, wherein

[0127] The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 35, and / or

[0128] The light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 36.

[0129] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises two identical heavy chains and two identical light chains, wherein

[0130] The heavy chain comprises a VH as described herein, or a HCDR1, HCDR2, and HCDR3 as described herein, and an Fc region and CH1 as described herein, or a heavy chain constant region as described herein;

[0131] The light chain comprises a VL described herein or LCDR1, LCDR2, and LCDR3 described herein, and a lambda light chain constant region comprising LLC160C and / or 166C described herein.

[0132] In a specific embodiment, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises two identical heavy chains and two identical light chains, wherein

[0133] The heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 35, and / or

[0134] The light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:37.

[0135] In a more specific embodiment, the anti-FRα antibody or antigen-binding fragment thereof of the invention comprises two identical heavy chains and two identical light chains, wherein

[0136] The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 35, and / or

[0137] The light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 37.

[0138] In some embodiments, the anti-FRα antibodies or antigen-binding fragments thereof of the present invention are chimeric or humanized antibodies.

[0139] In some embodiments, the antigen-binding fragment of an anti-FRα antibody of the invention is, for example, Fv, Fab, Fab', Fab'-SH, F(ab')2; dAb (domain antibody); a linear antibody; a single-chain antibody (e.g., scFv); a single-domain antibody such as VHH; a diabody or fragment thereof; or a camelid antibody.

[0140] In one embodiment, the anti-FRα antibody is a full-length antibody.

[0141] In one embodiment, the anti-FRα antibody also encompasses multispecific antibodies such as bispecific antibodies.

[0142] In some embodiments, the anti-FRα antibodies or antigen-binding fragments thereof of the present invention have good endocytic activity on FRα-positive cells, such as FRα-positive tumor cells.

[0143] In some embodiments, the present invention also provides a fusion protein comprising the anti-FRα antibody or antigen-binding fragment thereof of the present invention.

[0144] In some embodiments, the present invention also provides an immunoconjugate comprising an anti-FRα antibody or antigen-binding fragment thereof of the present invention, and further comprising a payload coupled to the antibody or antigen-binding fragment thereof. In some embodiments, the payload is a toxin, a small molecule drug / agent, a cytotoxic agent, an apoptotic agent, a chelating agent, an immunomodulator / immunostimulator / immunoagonist, an oligonucleotide, a polypeptide, a peptide epitope, a radionuclide, a prodrug, and the like. In some preferred embodiments, the payload is an immunostimulator / immunoactivator. In some more preferred embodiments, the payload is a TLR agonist. In some more preferred embodiments, the payload is a TLR7 / 8 agonist.

[0145] In some embodiments, the invention relates to an isolated nucleic acid encoding an anti-FRα antibody or antigen-binding fragment thereof of the invention.

[0146] In some embodiments, the present invention relates to a vector comprising the nucleic acid of the present invention, preferably the vector is an expression vector.

[0147] In some embodiments, the present invention relates to a host cell comprising a nucleic acid or vector of the present invention, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells (e.g., 293 cells or CHO cells, such as CHO-K cells or HEK293 cells) or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

[0148] In some embodiments, the present invention relates to a method for preparing an anti-FRα antibody or an antigen-binding fragment thereof, the method comprising

[0149] a) culturing the host cell of the present invention under conditions suitable for expressing the nucleic acid encoding the anti-FRα antibody or antigen-binding fragment thereof of the present invention,

[0150] b) optionally isolating said antibody or antigen-binding fragment thereof,

[0151] c) Optionally, the method further comprises recovering the anti-FRα antibody or antigen-binding fragment thereof from the host cell. Optionally, the antibody is purified, for example, by Protein A purification.

[0152] In another aspect, the present invention provides a novel TLR agonist, in particular a TLR7 / 8 agonist, which is selected from the following compounds or pharmaceutically acceptable salts or solvates thereof:

[0153] (1) Compound of formula (D-1):

[0154] in:

[0155] R1 is unsubstituted or substituted by one or more substituents selected from the group consisting of C1-C6 alkyl, hydroxy, halogen, cyano, C3-C6 cycloalkyl, amino, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C1-C6 alkoxy and C3-C6 cycloalkyloxy;

[0156] R2 and R3 are independently selected from hydrogen, C1-C6 alkyl, hydroxy, halogen, cyano, amino, C1-C6 alkoxy, aryl C1-C6 alkoxy and C1-C6 alkoxycarbonyl, wherein the aryl group is optionally substituted with one or more groups selected from the group consisting of C1-C6 alkyl, hydroxy, halogen, cyano, amino and C1-C6 alkoxy;

[0157] R4 is C1-C6 alkyl which is unsubstituted or substituted by one or more substituents selected from the group consisting of hydroxy, halogen, cyano, C3-C6 cycloalkyl, aryl C1-C6 alkyl, amino, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 alkoxy substituted by NHR5, and C1-C6 alkylcarbonyloxy substituted by NHR5; wherein the aryl group is optionally substituted by one or more groups selected from the group consisting of C1-C6 alkyl, hydroxy, halogen, cyano, amino, C1-C6 alkoxy, and C1-C6 alkylamino;

[0158] R5 is selected from C1-C6 alkyl and C1-C6 alkylcarbonyl, wherein said C1-C6 alkyl and C1-C6 alkylcarbonyl are optionally substituted by one or more substituents selected from the group consisting of hydroxy, halogen, amino, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C1-C6 alkoxy and C3-C6 cycloalkoxy;

[0159] (2) Compound of formula (D-2):

[0160] in:

[0161] X is absent or is -NH-(C=O)-, -(C=O)-NH- or C1-C6 alkylene, wherein the C1-C6 alkylene is optionally substituted with one or more substituents selected from hydroxy or halogen and one or more carbon atoms of the alkylene may be replaced by a group selected from -NH- and -(C=O)-;

[0162] X1 is selected from CH or N;

[0163] X2 is selected from CH2 or N;

[0164] X3 is NR8, wherein R8 is selected from H and C1-C6 alkyl, the C1-C6 alkyl optionally substituted with hydroxy, C1-C6 alkoxy and amino-substituted C1-C6 alkoxy;

[0165] X4 is CH2 or C(=O);

[0166] R6 and R7 are independently selected from H and C1-C6 alkyl, the C1-C6 alkyl being optionally substituted by one or more groups selected from hydroxy, halogen, cyano, C3-C6 cycloalkyl, amino, NH(C1-C6 alkyl), N(C1-C6 alkyl)2 and C1-C6 alkoxy;

[0167] and

[0168] represents a single bond or a double bond;

[0169] (3) Compound of formula (D-3):

[0170] in:

[0171] R9 is selected from hydroxy, amino, NH (C1-C6 alkyl), N (C1-C6 alkyl) 2 and C1-C6 alkoxy;

[0172] R 10 and R 11 independently selected from hydrogen and C1-C8 alkyl, said C1-C8 alkyl being optionally substituted with one or more selected from hydroxy, halogen, C1-C6 alkoxy and NR 15 R 16 substituted by a substituent, wherein R 15 and R 16 independently selected from H, C1-C6 acyl and hydroxy-substituted C1-C6 acyl;

[0173] R 12 is absent or is selected from hydrogen and arylC1-C6alkyl, wherein the aryl is optionally substituted with one or more substituents selected from hydroxy, halogen, C1-C6alkoxy and heterocyclylC1-C6alkyl, and the heterocyclyl is optionally substituted with one or more substituents selected from C1-C6alkyl, C1-C6alkoxy-C1-C6alkyl and C1-C6acyl;

[0174] R 13 is selected from hydrogen, halogen, hydroxyl or oxo (=O);

[0175] R 14 is selected from hydrogen, halogen, hydroxy, C1-C6 alkyl or C1-C6 alkoxy;

[0176] r is 0 or 1;

[0177] Y is selected from C or NR 17 , where R 17 is selected from hydrogen or C1-C6 alkyl optionally substituted by aryl or heterocyclic groups, wherein the aryl and heterocyclic groups are optionally selected from C1-C6 acyl, C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, hydroxy C1-C6 alkyl and NR 18 R 19 -C1-C6 alkyl substituted group, wherein R 18 and R 19 are independently selected from H, C1-C6 alkyl and C1-C6 acyl; and

[0178] Indicates a single bond or a double bond.

[0179] In one embodiment, the present invention provides a compound having a structure as shown in the above formula (D-1), which has a structure as shown in the formula (D-1'):

[0180] wherein R2 and R3 are as defined above; and

[0181] R is selected from H and

[0182] wherein R1′ and R2′ are independently selected from hydrogen and C1-C6 alkyl, and p′ is an integer selected from 1-6.

[0183] In one embodiment, the present invention provides a compound having a structure as shown in the above formula (D-2), wherein R6 and R7 are selected from C1-C6 alkyl, preferably n-propyl.

[0184] In one embodiment, the present invention provides a compound having a structure as shown in the above formula (D-3), which has a structure as shown in formula (D-3'):

[0185] in

[0186] R4' represents a C3-C6 alkyl group optionally substituted by one or more substituents selected from hydroxy, halogen, amino and C1-C6 alkoxy, preferably n-pentyl;

[0187] R5' is selected from H, C1-C6 alkyl, C1-C6 alkoxy-C1-C6 alkyl, C1-C6 acyl; preferably selected from H, acetyl, methoxyethyl.

[0188] In one embodiment, the present invention provides a compound having a structure as shown in the above formula (D-3), which has a structure as shown in formula (D-3"):

[0189] in:

[0190] Q is selected from OR6' and NR7'R8';

[0191] wherein R6' is selected from H and C1-C6 alkyl;

[0192] R7' and R8' are independently selected from H, C1-C6 acyl, C1-C6 acyl substituted with hydroxyl, preferably R7' and R8' are both H, or one of R7' and R8' is H and the other is acetyl or hydroxyacetyl.

[0193] In one embodiment, the present invention provides a compound having a structure as shown in the above formula (D-3), which has a structure as shown in the formula (D-3'):

[0194] in:

[0195] R9' is a C1-C6 alkyl group, preferably a C3-C6 straight-chain alkyl group, such as n-butyl;

[0196] r is 0 or 1;

[0197] X5 is selected from C1-C6 alkylene, for example -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-;

[0198] B is selected from aryl or heterocyclic groups, such as phenyl, tetrahydroisoquinolinyl and piperidinyl, and the aryl and heterocyclic groups are optionally substituted by C1-C6 acyl, C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, hydroxy C1-C6 alkyl or NR'R"-C1-C6 alkyl, and the R' and R" are independently selected from H, C1-C6 alkyl and C1-C6 acyl.

[0199] In one embodiment, the present invention provides a compound having a structure as shown in the above formula (D-1), which has a structure as shown in formula (D-1"):

[0200] wherein R1′ and R2′ are independently selected from hydrogen and C1-C6 alkyl, preferably R1′ and R2′ are both methyl, or R1′ is hydrogen and R2′ is 3,3-dimethyl-butyl.

[0201] In one embodiment, the present invention provides a compound as defined above, which is selected from the following compounds:

[0202] Preferably, the compound is selected from the following compounds:

[0203] In some embodiments, the compounds of the invention have balanced TLR7 and TLR8 stimulatory activity.

[0204] In a third aspect, the present invention also provides a TLR agonist connected to a linker, which is selected from a compound of formula (II), or a pharmaceutically acceptable salt or solvate thereof: L-(D) m (II)

[0205] in:

[0206] D is a TLR agonist as defined in the second aspect above;

[0207] L has the following structure: Z'-L 1- E-L2-L3-

[0208] Where L3 is connected to D;

[0209] Z' is selected from

[0210] L1 is -(CH2) n -C(=O)- or -(CH2) n -(O(CH2)2) t (CH2) n -C(=O)-;

[0211] E is absent or is a peptide residue comprising 2-10 amino acids, wherein the amino acid residue is a natural amino acid residue or a non-natural amino acid residue and is optionally substituted with a C1-C6 alkyl group, and the C-terminus of the peptide residue is covalently linked to L2;

[0212] L2 does not exist or

[0213] L3 does not exist or is -C(=O)-*, -C(=O)((CH2) n O) t (CH2) n -* or -NH(CH2)-*, wherein * indicates that the terminus is covalently linked to D;

[0214] n is an integer from 0 to 10,

[0215] t is an integer from 1 to 10,

[0216] m is an integer from 1 to 6.

[0217] In one embodiment, the present invention provides a compound of formula (II) as described above, or a pharmaceutically acceptable salt or solvate thereof, wherein

[0218] E has the structure shown below: -X1-X2-

[0219] Where X1 represents -(N(R 0 )C(R 0 )2C(=O)) s -, where R 0 independently of one another represent H or C1-C6 alkyl; s = 0-10, preferably 2, 4, 6 and 8;

[0220] X2 does not exist or is selected from

[0221] In one embodiment, the present invention provides a compound of formula (II) as described above, or a pharmaceutically acceptable salt or solvate thereof, wherein

[0222] E is selected from

[0223] Where s = 0-8.

[0224] In one embodiment, the present invention provides a compound of formula (II) as described above, or a pharmaceutically acceptable salt or solvate thereof, wherein

[0225] L- has a structure selected from the following:

[0226] In one embodiment, the present invention provides a compound of formula (II) as described above, or a pharmaceutically acceptable salt or solvate thereof, selected from:

[0227] In one embodiment, the compound of formula (II) is selected from the following compounds

[0228] In some embodiments, the present invention also provides an immunoconjugate comprising a targeting moiety, a TLR agonist of the present invention, and / or a linker of the present invention for connecting a targeting moiety to a TLR agonist. In some embodiments, the targeting moiety is a specific antigen binding protein or a fragment thereof, for example, a molecule with an antibody or immunoglobulin structure, a free receptor, a cyclic peptide, etc. In some preferred embodiments, the targeting moiety is an antibody or an antigen binding fragment thereof. In some preferred embodiments, the targeting moiety (e.g., an antibody) causes and / or promotes the activation of immune cells, for example, by ADCP.

[0229] In some preferred embodiments, the immunoconjugates of the present invention comprise a TLR agonist (e.g., a TLR7 / 8 agonist of the present invention as defined above), preferably coupled to a tumor-targeting antibody. The immunoconjugates of the present invention are well tolerated in vivo when systemically administered and induce strong activation of intratumoral myeloid cells, leading to tumor clearance and subsequent immune memory.

[0230] In some embodiments, the present invention provides an immunoconjugate having a structure as shown in formula (I), Ab-(L-(D) m ) p (I)

[0231] or a pharmaceutically acceptable salt or solvate thereof,

[0232] in:

[0233] Ab is a specific antigen binding protein or a fragment thereof, preferably a monoclonal antibody or a fragment thereof;

[0234] L is a linker;

[0235] D is a TLR agonist, preferably a TLR7 / 8 agonist; and

[0236] m is an integer from 1 to 6; and

[0237] p is a value from 1 to 16, for example 2 to 10. In some embodiments, D in formula (I) is a compound as defined in the second aspect of the present invention.

[0238] In some embodiments, LD in formula (I) is a compound of formula (II) as defined in the third aspect of the present invention.

[0239] In some embodiments, -L- in formula (I) has a structure selected from the group consisting of:

[0240] The left side of the structure is connected to Ab, and the right side is connected to D.

[0241] In some embodiments, L-(D) in formula (I) m Having a structure selected from the following:

[0242] In some embodiments, the antibody immunoagonist conjugate of the present invention, or a pharmaceutically acceptable salt or solvate thereof, has a DAR value of 1-10, such as 2-8.

[0243] In some embodiments, the antibody immunoagonist conjugate is selected from

[0244] wherein Ab is an antibody or antigen-binding fragment thereof that binds to TROP2 (eg, human TROP2) or FRα (eg, human FRα), preferably as defined above; q represents DAR, eg, 1-10, eg, 2-8, eg, about 3, 4, 5, or 6.

[0245] In some embodiments, Ab in formula (I) is a specific antigen binding protein or a fragment thereof. In some embodiments, Ab is an antibody. In some embodiments, Ab is an antibody fragment. In some embodiments, Ab is a monoclonal antibody or a fragment thereof. In some preferred embodiments, Ab causes and / or promotes the activation of immune cells, for example, through ADCP action. In some preferred embodiments, Ab specifically binds to TROP2. In some preferred embodiments, Ab specifically binds to FRα. In some preferred embodiments, Ab has a cysteine ​​mutation in its constant region.

[0246] In some embodiments, the antibody immunoagonist conjugate of formula (I) or its subformula, or a composition comprising one or more of said antibody immunoagonist conjugates (e.g., TROP2-ISAC or FRα-ISAC) of the present invention has an average DAR of any value selected from the following ranges: 1±0.4, 2±0.4, 3±0.4 or 4±0.4.

[0247] In some preferred embodiments, Ab in formula (I) is an antibody or antigen-binding fragment thereof that specifically binds to TROP2. In some preferred embodiments, the antibody that specifically binds to TROP2 binds to a different epitope than the TROP2 cytotoxic ADC.

[0248] In some preferred embodiments, Ab in formula (I) is an antibody or antigen-binding fragment thereof that specifically binds to FRα. In some preferred embodiments, the antibody that specifically binds to FRα binds to a different epitope than the FRα cytotoxic ADC.

[0249] In some embodiments, in an antibody immunoagonist conjugate comprising an anti-FRα antibody or antigen-binding fragment thereof of the present invention, the cysteine ​​mutation allows the anti-FRα antibody or antigen-binding fragment thereof to be coupled to a linker via the mutated cysteine, for example, to achieve site-specific coupling of the immunoagonist.

[0250] In another aspect, the present invention provides a pharmaceutical composition comprising an antibody or fragment thereof, nucleic acid, vector, host cell, fusion protein, immunoconjugate or pharmaceutically acceptable salt or solvate thereof as described above, and optionally one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators, and optional pharmaceutical excipients. In another aspect, the present invention provides a pharmaceutical combination comprising an antibody or fragment thereof, nucleic acid, vector, host cell, fusion protein, immunoconjugate or pharmaceutically acceptable salt or solvate thereof, or pharmaceutical composition as described above, and one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.

[0251] In another aspect, the present invention provides a method for preventing or treating a tumor in a subject, comprising administering to the subject an effective amount of the antibody or fragment thereof, nucleic acid, vector, host cell, fusion protein, immunoconjugate or pharmaceutically acceptable salt or solvate thereof as described above, or the pharmaceutical composition as described above, or the pharmaceutical combination as described above.

[0252] In another aspect, the present invention provides the use of the antibody or fragment thereof, nucleic acid, vector, host cell, fusion protein, immunoconjugate or pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition as described above, or the drug combination as described above, for preparing an anti-tumor agent.

[0253] In some embodiments, the tumor is a cancer, preferably, the cancer has elevated levels (eg, nucleic acid or protein levels) of TROP2 or FRα, eg, compared to healthy individuals or healthy tissue adjacent to the patient's cancer tissue.

[0254] In some embodiments, the cancer is selected from lung cancer, melanoma, head and neck tumors, prostate cancer, esophageal cancer, cervical cancer, kidney cancer, bladder cancer, ovarian cancer, pancreatic cancer, or breast cancer.

[0255] In some embodiments, the method further comprises administering to the patient one or more therapies, such as therapeutic modalities and / or other therapeutic agents, preferably, the therapeutic modalities comprise radiation therapy or surgery, or the therapeutic agents comprise chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators.

[0256] The anti-TROP2 antibodies and / or immunoconjugates of the present invention have at least the following advantages:

[0257] (1) Binds to human and cynomolgus monkey TROP2 with high affinity;

[0258] (2) has excellent anti-tumor efficacy in vivo;

[0259] (3) Good safety in animal experiments, such as in mice and monkeys;

[0260] and

[0261] (4) Compared with other immunoconjugates, such as TROP2 ADC, it has non-competitive epitopes, which is conducive to combination / combination therapy.

[0262] The immunoconjugates of the present invention have the following advantages in particular:

[0263] (1) As a TROP2 ISAC, it has more significant single-agent efficacy compared with other ISACs with similar efficacy and has good safety in mice and monkeys.

[0264] (2) Use polysarcosine / polyethylene glycol (PEG) as a stable linker to avoid the high toxicity of the released payload TLR7 / 8 agonist and avoid the immunogenicity of the PEG linker

[0265] (3) As monotherapy, the TROP2-TLR7 / 8 agonist ISAC showed potent antitumor activity in the treatment of multiple TROP2+ heterologous tumor models, including, for example, gastric cancer and pancreatic cancer.

[0266] The anti-FRα antibodies and / or immunoconjugates of the present invention have at least the following advantages:

[0267] (1) Binds to human, cynomolgus monkey, and mouse FRα with high affinity;

[0268] (2) has excellent anti-tumor efficacy in vivo;

[0269] (3) Good safety in animal experiments, such as in mice;

[0270] and

[0271] (4) Compared with other immunoconjugates, such as FRαADC, it has non-competitive epitopes, which is conducive to combination / combination therapy.

[0272] The immunoconjugates of the present invention have the following advantages in particular:

[0273] (1) As a FRα ISAC, it has more significant single-agent efficacy compared with other ISACs with similar efficacy and has good safety in mice.

[0274] (2) Use polysarcosine / polyethylene glycol (PEG) as a stable linker to avoid the high toxicity of the released payload TLR7 / 8 agonist and avoid the immunogenicity of the PEG linker.

[0275] (3) As monotherapy, the FRα-TLR7 / 8 agonist ISAC showed potent antitumor activity in the treatment of FRα+ heterologous tumor models, such as various types of ovarian cancer.

[0276] (4) The immune agonist is linked to the antibody via the mutated cysteine ​​site, thereby

[0277] (i) The conjugate is more hydrophilic,

[0278] (ii) The conjugate is more stable in plasma, and small molecule toxins are less likely to be shed;

[0279] (iii) The conjugate has stronger efficacy in animals. For example, an ISAC with an average DAR of 4 can have the same efficacy as an antibody conjugate with a DAR of 8 that has not been modified with cysteine ​​mutations. Description of the drawings:

[0280] FIG1A to FIG1C show the results of antibody-dependent cellular phagocytosis (ADCP) functional assays.

[0281] FIG2 shows the results of antibody-dependent cellular phagocytosis (ADCP) functional assay of the humanized antibody.

[0282] FIG3 shows the macrophage endocytosis function test results of humanized antibodies.

[0283] Figures 4A to 4D show the activity test results of the compounds of the present invention in H-TLR7 NFKB Reporter 293 and H-TLR8 NFKB Reporter 293 cell lines, wherein Figures 4A and 4B show the TLR7 activity of each compound, and Figures 4C and 4D show the TLR8 activity of each compound.

[0284] FIG5 shows the detection results of the TROP2-ISAC of the present invention in a PBMC and tumor cell co-culture system.

[0285] Figures 6A to 6D show the anti-tumor effect of TROP2-ISAC of the present invention in the NCI-N87 model, wherein Figures 6A and 6C show the changes in tumor volume over time, and Figures 6B and 6D show the changes in animal body weight over time.

[0286] Figures 7A and 7B show the anti-tumor effect of TROP2-ISAC of the present invention in the BxPC3 model, wherein Figure 7A shows the changes in tumor volume over time, and Figure 7B shows the changes in animal body weight over time.

[0287] 8A and 8B show the results of toxicity experiments of TROP2-ISAC of the present invention in BALB / C mice.

[0288] FIG9 shows the results of the toxicity experiment of TROP2-ISAC of the present invention in hTROP2 KI mice.

[0289] Figures 10A to 10C show the anti-tumor effect of TROP2-ISAC of the present invention in the CT26-hTROP2 model, wherein Figure 10A shows the changes in tumor volume over time, Figure 10B shows the changes in animal body weight over time, and Figure 10C shows the changes in animal survival rate over time.

[0290] Figures 11A and 11B show the anti-tumor effect of the TROP2-ISAC combined with TROP2-ADC of the present invention in the BxPC3 model, wherein Figure 11A shows the changes in tumor volume over time, and Figure 11B shows the changes in animal body weight over time.

[0291] Figures 12A and 12B show the anti-tumor effect of the TROP2-ISAC combined with TROP2-ADC of the present invention in the LK-2 model, wherein Figure 12A shows the changes in tumor volume over time, and Figure 12B shows the changes in animal body weight over time.

[0292] 13A and 13B show the results of high-dose toxicity experiments of TROP2-ISAC of the present invention in BALB / C mice.

[0293] FIG14 shows the ADCP activity of the anti-FRα antibody clone BC1254-A1, compared with other antibodies.

[0294] FIG15 shows the macrophage ADCP activity of the anti-FRα antibody clone BC1254-A1.

[0295] FIG16 shows the average DAR values ​​of BC1254-A1 V2-Compound 6 conjugates determined by RP-HPLC.

[0296] FIG17 shows the average DAR values ​​of BC1254-A1-Compound 6 conjugates determined by HIC-HPLC.

[0297] FIG18 shows the comparison of ADCP activities of the anti-FRα antibody clone BC1254-A1 before and after cysteine ​​mutation modification and before and after coupling with an immune activator.

[0298] FIG19 shows the evaluation of the immunostimulatory effect of anti-FRα-ISAC in a co-culture system with PBMCs and target tumor cells by measuring TNFα secretion.

[0299] FIG20 shows the evaluation of the immunostimulatory effect of anti-FRα-ISAC in a co-culture system with PBMCs and target tumor cells by measuring IFNα secretion.

[0300] Figures 21A and 21B are line graphs showing the changes in tumor volume and mouse body weight over time in model mice bearing subcutaneous OV90 cell tumors after FRα-ISAC treatment.

[0301] Figures 22A and 22B are line graphs showing the changes in tumor volume and mouse body weight over time in model mice bearing subcutaneous tumors of A2780cisR-FRα cells after treatment with FRα-ISAC.

[0302] Figures 23A and 23B show line graphs showing the changes in tumor volume and mouse body weight over time in model mice bearing subcutaneous tumors of A2780cisR-FRα cells after combined treatment with FRα-ISAC and FRα-ADC.

[0303] FIG24 shows the toxicity of FRα-ISAC to mice measured by observing the changes in body temperature of mice.

[0304] Detailed Description of the Invention

[0305] I. Definition

[0306] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0307] To interpret this specification, the following definitions will apply, and wherever 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 limiting.

[0308] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within 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.

[0309] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0310] As used herein, the terms "comprising" or "including" are intended to include the recited elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, combinations of the recited elements, integers, or steps are also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0311] The terms "TROP2" and "TACD2" are used interchangeably herein and refer to a human trophoblast cell surface glycoprotein antigen. It is a type I cell surface glycoprotein that is highly expressed in human cancers. It was originally identified as an antigen present on human gastrointestinal tumors and is the second of two members of this family. The sequences of mouse and cynomolgus macaque TROP2 have high homology to their human counterparts. In some embodiments of the present invention, TROP2 is human TROP2. In some embodiments, TROP2 is a protein under UniProt database accession number P09758.

[0312] As used herein, the terms "anti-TROP2 antibody," "anti-TROP2," "TROP2 antibody," or "anti-TROP2 antibody" refer to antibodies that bind to the TROP2 protein with sufficient affinity. The antibodies can be used as diagnostic and / or therapeutic agents targeting TROP2, or used to construct immunoconjugates, such as antibody-immunoagonist conjugates.

[0313] The terms "FOLR1" and "FRα" are used interchangeably herein and refer to folate receptor alpha (FRα), also known as folate receptor 1 (FOLR1), a member of the folate receptor family. It is a cell surface glycoprotein with a molecular weight of 38-40 kD. FRα expression levels are significantly increased in certain rapidly proliferating cancer cells. In some embodiments of the present invention, FRα is human FRα. In some embodiments, FRα is the protein identified in the UniProt database under accession number P15328.

[0314] As used herein, the terms "anti-FRα antibody," "anti-FRα," "FRα antibody," or "FRα-targeting antibody" refer to an antibody or antigen-binding fragment thereof that binds to the FRα protein with sufficient affinity. The antibody can be used as a diagnostic and / or therapeutic agent targeting FRα, or used to construct an immunoconjugate, such as an antibody-immunoagonist conjugate.

[0315] The term "antigen" refers to a molecule that triggers an immune response. This immune response may involve the production of antibodies or the activation of specific immune cells, or both. It will be appreciated by those skilled in the art that any macromolecule, including substantially all proteins or peptides, can be used as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to a portion of an antigen (e.g., TROP2) that specifically interacts with an antibody molecule.

[0316] The terms "complete antibody", "whole antibody" or "full-length antibody" are used interchangeably herein and refer to antibody molecules with the structure of natural immunoglobulin molecules. In the case of conventional four-chain IgG antibodies, the full-length antibody comprises two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of heavy chain antibodies having only heavy chains and lacking light chains, the full-length antibody comprises two heavy chains (H) interconnected by disulfide bonds. For conventional four-chain IgG antibodies, the full-length antibody heavy chain is generally composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, wherein the heavy chain constant region comprises at least three domains CH1, CH2 and CH3. The full-length antibody light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region VH and each light chain variable region are composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0317] The term "antigen-binding fragment" of an antibody is a molecule that is different from a full-length antibody and that contains a portion of a full-length antibody, but that 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 antigen binding. Antigen-binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, dAb (domain antibody), linear antibodies, single-chain antibodies (e.g., scFv); single-domain antibodies such as VHH, diabodies or fragments thereof, or camelid antibodies, diabodies, single-domain antibodies (sdAb), and nanobodies. For example, Fab fragments can be obtained by digesting a full-length antibody with papain. In addition, digesting a full antibody below the disulfide bonds in the hinge region with pepsin produces F(ab')2, which is a dimer of Fab' and a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by destroying the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer. A Fab' monomer is essentially a Fab fragment with a hinge region. An Fv fragment consists of the VL and VH domains of a single antibody arm. The two domains of the Fv fragment, VL and VH, can be encoded by separate genes, but recombinant methods can also be used to connect the two domains using a synthetic linker peptide to produce them as a single protein chain, in which the VL and VH regions are paired to form a single-chain Fv (scFv).

[0318] The term "single-chain antibody (scAb)" is used herein in the broadest sense and specifically covers antibodies with monospecificity or multispecificity (e.g., bispecificity) that are initially produced as a single continuous polypeptide chain. Such single-chain antibodies include, but are not limited to, antibodies having two linked VL and VH regions. In one embodiment, the single-chain antibody is an scFv.

[0319] "Diabodies" are small, bivalent antibodies constructed through gene fusion, for example, dimers composed of two polypeptide chains. The VL and VH domains of each polypeptide chain of a diabody are linked by a linker, so that the VL and VH encoded in the same polypeptide chain form a dimer with different single-chain variable region segments. Diabodies generally have two antigen-binding sites.

[0320] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of antibodies 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 Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and North's CDR definitions based on affinity propagation clustering using a large number of crystal structures (North et al., "A New Clustering of Antibody CDR Loop Concepts", Journal of Molecular Biology, 406, 228-256 (2011)).

[0321] The following are the regional ranges of CDRs defined using the Kabat, AbM, Chothia, Contact, and IMGT schemes.

[0322] A CDR can also be identified based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention).

[0323] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-mentioned ways.

[0324] Unless otherwise indicated, in the present invention, when referring to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues), the numbering refers to the position 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)).

[0325] In one embodiment, the heavy chain variable region CDRs of the antibodies of the present invention are determined according to the following rules:

[0326] VH CDR1 was determined according to the Kabat & Chothia rules; and VH CDR2 and 3 were both determined according to the Kabat rules.

[0327] In one embodiment, the light chain variable region CDRs of an antibody of the invention are determined according to the Kabat rules.

[0328] In one embodiment, the heavy chain variable region CDRs of the antibodies of the present invention are determined according to the following rules: VH CDR1 is determined according to the Kabat & Chothia rules; and VH CDR2 and 3 are both determined according to the Kabat rules; and the light chain variable region CDRs are determined according to the Kabat rules.

[0329] It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems may be different. Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0330] An "antibody that binds to the same or overlapping epitope as a reference antibody" refers to an antibody that blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the reference antibody to its antigen in a competition assay, whereas conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the antibody to its antigen in a competition assay. Accordingly, if two antibodies or derivatives thereof bind to non-competing epitopes, they bind to non-identical or non-overlapping epitopes and preferably do not exhibit mutual blocking and / or interference with antigen binding in a competition assay. Numerous types of competitive binding assays can be used to determine whether one antibody competes with another, such as solid phase direct or indirect radioimmunoassays (RIAs), solid phase direct or indirect enzyme immunoassays (EIAs), and sandwich competition assays.

[0331] An antibody that exhibits the same or similar binding affinity and / or specificity as a reference antibody is an antibody that has at least 50%, 60%, 70%, 80%, 90% or more than 95% of the binding affinity and / or specificity of the reference antibody. This can be determined by any method known in the art for determining binding affinity and / or specificity.

[0332] The term "chimeric antibody" is an antibody molecule in which (a) the constant region or a portion thereof is changed, replaced or exchanged so that the antigen binding site is connected to a constant region of a different or altered class, effector function and / or species or a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) that imparts new properties to the chimeric antibody; or (b) the variable region or a portion thereof is changed, replaced or exchanged with a variable region having a different or altered antigenic specificity. For example, a mouse 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 the antigen while having reduced immunogenicity in humans as compared to the original mouse antibody.

[0333] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (e.g., a mouse monoclonal antibody) while being less immunogenic when administered to humans, for example, as a therapeutic. 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 portions of the variable region not involved in binding with the corresponding portions of a human antibody).

[0334] The term "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" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a native antibody, the immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD class antibodies; or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE class antibodies. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or heavy chain constant region is according to the EU numbering system (also called the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Herein, the term "Fc region" does not include the heavy chain variable region VH and light chain variable region VL and the heavy chain constant region CH1 and light chain constant region CL of an immunoglobulin, but may include the hinge region at the N-terminus of the heavy chain constant region in some cases.

[0335] The term "amino acid substitution" or "amino acid mutation" refers to the replacement of at least one amino acid residue in a predetermined parent amino acid sequence with a different "substituted" amino acid residue. The replacement residue or residues can be "naturally occurring amino acid residues" (i.e., encoded by the genetic code) and are selected from the group consisting of: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine ​​(Cys); glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (Ile); leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val).

[0336] The amino acid position to be mutated to cysteine ​​is generally indicated by "chain type, mutation position." In this article, unless otherwise specified, LLC represents lambda light chain, LC represents kappa light chain, and HC represents heavy chain. Thus, "LLC160C" means that the amino acid at EU position 160 of the lambda light chain is substituted with cysteine ​​(C).

[0337] When referring to heavy chain amino acid positions in the present invention, unless otherwise specified, it refers to the amino acid positions numbered according to the IgG1 heavy chain, that is, it covers the amino acid positions numbered based on the IgG1 heavy chain and the amino acid positions corresponding to the amino acid positions on other heavy chains.

[0338] "Sequence identity" is defined as the percentage of identical residues in amino acid sequence variants after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.

[0339] "Parent protein (e.g., parent antibody or parent constant region or parent Fc region)" refers to a protein comprising an amino acid sequence in which one or more amino acid residues are to be replaced with one or more other amino acid residues, such as cysteine ​​residues. The parent protein may comprise a native or wild-type sequence. The parent protein may have existing amino acid sequence modifications (such as additions, deletions, and / or substitutions) relative to other native, wild-type, or modified forms of the protein. The parent antibody may be directed against a target antigen of interest, such as a biologically important polypeptide.

[0340] As used herein, the term "binding" or "specific binding" means that the binding effect is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as by radioimmunoassay (RIA) or thin-layer interferometry or MSD assays or surface plasmon resonance (SPR).

[0341] "Immunoconjugate" in this article refers to a payload connected to an antibody or its antigen-binding fragment by a linker, so that the antibody or its antigen-binding fragment can be used as a carrier to target and transport the payload to a target site. The term "payload" refers to the active portion of the antibody or antibody fragment of the present invention that is conjugated, and can include any portion for attaching the antibody or antibody fragment. In some embodiments, the payload can be a drug such as a small molecule drug, a radionuclide, DNA, RNA, an enzyme or a polypeptide. In some embodiments, immunoconjugates encompass antibody-drug conjugates (ADC), antibody immunostimulatory agent (agonist) drug conjugates (ISAC), antibody oligonucleotide conjugates (AOC), antibody polypeptide drug conjugates (APC), antibody nuclide drug conjugates (RDC) or antibody degradation drug conjugates (ADeC) and the like. Suitable payloads or active moieties for attachment to antibodies include, for example, cytotoxic agents, chemotherapeutic agents, innate immune agonists (e.g., Toll-like receptor agonist (TLR) ISAC drugs SBT6050, SBT6290, BDC-1001; STING agonist ISAC drug XMT-2056, Treg cell regulation ISAC drug ADCT-301, etc.), immunomodulators, therapeutic oligonucleotides (siRNA, PMO, etc.), or radionuclides, etc. In some embodiments, the immunoconjugate of the present invention is an antibody-immunoagonist conjugate, i.e., an ISAC.

[0342] As used herein, "antibody immunoagonist conjugate (ISAC)" refers to a structure obtained by linking an antibody to an immunoagonist (preferably a TLR7 / 8 agonist).

[0343] The term "linker" refers to a structural fragment that connects a drug (e.g., a small molecule drug such as a TLR7 / 8 agonist) to an antibody portion. It should be understood that the linker has a functional group that can form a bond with a functional group of the antibody or antigen-binding fragment thereof before being attached to the antibody or antigen-binding fragment thereof.

[0344] The term "linker-payload" refers to a compound formed by linking a payload, such as a drug (eg, a small molecule drug such as a TLR7 / 8 agonist), to a linker.

[0345] The term "site-specific conjugation" as used herein refers to conjugation in which a drug is specifically linked to a specific site of an antibody via a linker.

[0346] Term " alkyl " as herein described refers to fully saturated branched or unbranched hydrocarbon group.Alkyl preferably comprises 1-16 carbon atoms, for example 1-12 carbon atoms, 1-10 carbon atoms, 1-6 carbon atoms or 1-4 carbon atoms.For example, C1-C6 alkyl refers to fully saturated branched or unbranched hydrocarbon group comprising 1-6 carbon atoms.Representative examples of alkyl include but are not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl etc.

[0347] The term "C1-C6 alkoxy" as used herein refers to a group of the formula -O-(C1-C6 alkyl), wherein alkyl is as defined above. Representative examples of C1-C6 alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, and tert-butoxy.

[0348] The term "C1-C6 acyl" as used herein refers to a group of the formula -C(=O)-(C1-C6 alkyl), wherein alkyl is as defined above. Representative examples of C1-C6 acyl include, but are not limited to, formyl, acetyl, propionyl, and the like.

[0349] The term "heterocyclyl" as used herein refers to a monovalent saturated or partially unsaturated monocyclic or bicyclic ring system having 3 to 10 ring atoms, containing 1 to 5 ring heteroatoms selected from N, O and S, with the remaining ring atoms being carbon. Representative examples of heterocyclyl include, but are not limited to, aziridinyl, oxirane, azetidinyl, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, pyrazolidinyl, imidazolidinyl, piperidinyl, tetrahydropyranyl, piperazinyl, morpholinyl, thiomorpholinyl, dioxothiomorpholinyl, azepanyl, diazepanyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

[0350] The term "aryl" refers to a monocyclic or condensed-ring aromatic hydrocarbon group having a conjugated π electron system, such as phenyl and naphthyl.

[0351] The term "alkenyl" refers to a straight or branched chain hydrocarbon group containing 2 to 16 carbon atoms and including at least one double bond and no triple bonds. Alkenyl groups preferably contain 2 to 12 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms, 2 to 6 carbon atoms, or 2 to 4 carbon atoms. Representative examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, and the like.

[0352] The term "alkynyl" refers to a straight or branched chain hydrocarbon group containing 2 to 16 carbon atoms and at least one triple bond. Alkynyl groups preferably contain 2 to 12 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms, 2 to 6 carbon atoms, or 2 to 4 carbon atoms. Representative examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.

[0353] The term "halogen" or "halo" refers to fluorine (-F), chlorine (-Cl), bromine (-Br), and iodine (-I).

[0354] The term "haloalkyl" refers to an alkyl group as defined herein that is substituted with one or more halogen groups as defined herein. Halogenated alkyl groups may preferably be monohalogenated alkyl, dihalogenated alkyl, or polyhalogenated alkyl (including perhalogenated alkyl). Monohalogenated alkyl groups may contain one iodine, bromine, chlorine, or fluorine in the alkyl group. Dihalogenated alkyl and polyhalogenated alkyl groups may contain two or more identical halogen atoms or a combination of different halo groups in the alkyl group. Preferably, polyhalogenated alkyl groups contain up to 12, 10, 8, 6, 4, 3, or 2 halogen groups. Non-limiting examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl. Perhalogenated alkyl groups refer to an alkyl group in which all hydrogen atoms are replaced by halogen atoms.

[0355] The term "haloalkenyl" refers to an alkenyl group, as defined herein, substituted with one or more halo groups, as defined herein. The term "haloalkynyl" refers to an alkynyl group, as defined herein, substituted with one or more halo groups, as defined herein. The meaning of "halo" as defined for "haloalkyl" applies to both "haloalkenyl" and "haloalkynyl."

[0356] The term "polyol group" refers to an alkyl group as defined above containing a plurality (e.g. 2-10, e.g. 3, 4, 5, 6, 7 or 8) of hydroxyl groups, which optionally contains 1 or more (e.g. 2, 3 or 4) other groups (e.g. amino, carbonyl). Non-limiting examples of "polyol groups" include, for example

[0357] The term "amino acid" refers to naturally occurring and synthetic amino acids. Amino acids can be L or D isomers. The compilation of conventional amino acids referred to herein follows conventional usage. See, for example, Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. And in this disclosure, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, glycine can be represented by Gly, alanine can be represented by Ala, valine can be represented by Val, glutamine can be represented by Gln, glutamic acid can be represented by Glu, phenylalanine can be represented by Phe, and leucine can be represented by Leu.

[0358] The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur. For example, when a group or structure is "optionally substituted," the group or structure may be substituted or unsubstituted.

[0359] The term "pharmaceutically acceptable salt" refers to salts that retain the biological effects and properties of the ISAC conjugates of the present invention and are not biologically or otherwise undesirable. The ISAC conjugates of the present invention may exist as pharmaceutically acceptable salts thereof, including acid addition salts and base addition salts. In the present invention, pharmaceutically acceptable, non-toxic acid addition salts refer to salts formed between the ISAC conjugates of the present invention and organic or inorganic acids, including but not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, and the like. Pharmaceutically acceptable non-toxic base addition salts refer to salts formed by the ISAC conjugates of the present invention with organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; organic base salts, such as ammonium salts formed with organic bases containing N groups.

[0360] The term "solvate" refers to an association formed between one or more solvent molecules and the antibody immunoagonist conjugate of the present invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, and the like.

[0361] As used herein, "pharmaceutically acceptable" and "pharmaceutically acceptable" are used interchangeably unless there is any contradiction in the context.

[0362] The term "drug:antibody ratio" or "DAR" refers to the average ratio of the cargo moiety (D) to the Ab moiety conjugated to an immunoconjugate (e.g., ISAC) described herein. In other words, the DAR value reflects the average number of cargo molecules carried by each ISAC molecule based on a single antibody molecule (Ab moiety) in a population of immunoconjugates.

[0363] In some embodiments described herein, a majority of the ISAC molecules in a population of immunoconjugates carry the same number of cargo molecules, in which case the DAR can be approximately determined by m and p in formula (I), for example, the DAR can be 1 to 16, for example, 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The DAR can also be determined by the overall ratio of drug moiety (D) conjugated to the Ab moiety described herein to the Ab moiety in the product as measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis, and / or HPLC), for example, when the number of cargo molecules (e.g., m×p) carried by individual ISAC molecules in a population of immunoconjugates varies widely. In some embodiments, the DAR value of the conjugate of the invention is 1 to 16, e.g., 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, e.g., 1.0-8.0, 2.0-6.0, e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, and ranges having two of these values ​​as endpoints.

[0364] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immunosuppressants).

[0365] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0366] "Chemotherapeutic agents" include chemical compounds useful in treating cancer or immune system disorders.

[0367] The term "drug" refers to an organic compound capable of modulating biological processes, particularly altering or preventing pathological processes.

[0368] The term "prodrug" refers to a chemically modified active or inactive compound that, after administration to a subject, undergoes physiological actions in the body (e.g., hydrolysis, necrolysis, etc.) to become an active drug. The techniques for making and using prodrugs are well known to those skilled in the art.

[0369] The term "small molecule drug" refers to low molecular weight organic compounds that can modulate biological processes, particularly alter or prevent pathological processes. A "small molecule" is defined as a molecule with a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 1 kD, more preferably less than 500 kD. Small molecule drugs include, but are not limited to, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimics. As therapeutic agents, small molecules can be more cell-permeable, less susceptible to degradation, and less prone to eliciting an immune response than larger molecules.

[0370] "Anti-tumor compounds" are pharmaceutically active compounds that have an effect on tumors, including but not limited to cytotoxic agents or chemotherapeutic agents, such as the cytotoxic agents disclosed in WO2021 / 173773, camptothecin compounds Exitecan (topoisomerase I inhibitor Exatecan), DXd (a new topoisomerase I inhibitor Exatecan derivative), auristatin compounds such as monomethyl auristatin E (MMAE) or maytansine compounds such as small molecule microtubule inhibitor DM1. It should be understood that anti-tumor compounds can be substituted by isotopes including but not limited to deuterium, tritium, etc. For example, after substitution with deuterium, the carbon-deuterium bond replaces the carbon-hydrogen bond. Since the former is more stable than the latter, the substitution can directly affect the absorption, distribution, metabolism and excretion properties of certain drugs, thereby improving the efficacy, safety and tolerability of the drug. Therefore, the "anti-tumor compound" of the present application can cover compounds substituted by deuterium.

[0371] "Deuterium-substituted" means that a hydrogen in the molecule is replaced by deuterium, eg, 1 or more hydrogens, eg, 1-10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) are replaced by deuterium.

[0372] The term "immunomodulator" as used herein refers to a natural or synthetic agent or drug that inhibits or regulates (e.g., activates) an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include immunosuppressants. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0373] The term "effective amount" refers to an amount or dosage of an antibody, fragment, composition, or combination of the present invention that, after administration to a patient in a single or multiple doses, produces the desired effect in a patient in need of treatment or prevention. Depending on the desired effect, both a "therapeutically effective amount" and a "prophylactically effective amount" may be included.

[0374] A "therapeutically effective amount" is an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 30%, even more preferably by at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100% relative to an untreated subject.

[0375] A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result, at the required dosage and for the required period of time. Typically, a prophylactic amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0376] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny screened or selected for the same function or biological activity as the initially transformed cell are included herein.

[0377] The term "label" as used herein 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 promotes the detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or can catalyze a chemical change in a detectable substrate compound or composition in the case of an enzymatic label. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically connecting) a detectable substance to the probe or antibody and indirect labeling of the probe or antibody by reacting with another reagent of the direct label.

[0378] "Individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, 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.

[0379] An "isolated" antibody or other molecule (e.g., ISAC molecule) is one that has been separated from a component of its natural environment or the environment in which it is expressed. In some embodiments, the antibody or ISAC 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).

[0380] 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 tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0381] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Cancer can be in the early, middle, or late stages or be metastatic. Cancers suitable for treatment by the molecules of the invention include, but are not limited to, lung cancer, melanoma, head and neck tumors, prostate cancer, esophageal cancer, cervical cancer, kidney cancer, bladder cancer, ovarian cancer, pancreatic cancer, or breast cancer, including metastatic forms of those cancers.

[0382] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. "Tumor" encompasses solid tumors and hematological tumors, as well as metastatic lesions. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein.

[0383] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0384] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0385] The term "pharmaceutical combination" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit and a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) an antibody and / or ISAC molecule of the present invention, and (ii) an additional therapeutic agent) are administered to a patient as separate entities simultaneously, without specific time restrictions, or sequentially at equal or different time intervals, wherein such administration provides prophylactically or therapeutically effective levels of the two or more active agents in the patient. In some embodiments, the antibody and / or ISAC molecule of the present invention and the additional therapeutic agent used in the pharmaceutical combination are administered at levels no greater than when they are administered alone. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously as a single entity. The dosages and / or time intervals of the two or more active agents are preferably selected so that the combined use of the components produces an effect greater than that achieved by either component alone in treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0386] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein. This administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, this administration includes co-administration of each active ingredient in a variety of or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids can be reconstituted or diluted to the desired dose before administration. In addition, this 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 therapeutic regimen will provide the beneficial effects of the drug combination in treating disorders or conditions described herein.

[0387] As used herein, "treat," ...

[0388] As used herein, "prevention" includes the inhibition of the development or progression of a disease or condition, or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the development of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0389] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0390] "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 an organ or tissue sample or a biopsy sample or a puncture sample from a fresh, frozen and / or preserved organ; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during the subject's pregnancy or development. Tissue samples may contain compounds that are not naturally contaminated with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0391] II. Antibodies, Nucleic Acids, and Host Cells

[0392] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof of the present invention comprises three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3.

[0393] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof of the present invention comprises three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0394] In some embodiments, the anti-TROP2 antibody or antigen-binding fragment thereof of the present invention comprises three complementarity determining regions (HCDRs) from a heavy chain variable region and three complementarity determining regions (LCDRs) from a light chain variable region.

[0395] In some aspects, the anti-TROP2 antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH). In some aspects, the anti-TROP2 antibodies or antigen-binding fragments thereof of the present invention comprise a light chain variable region (VH). In some aspects, the anti-TROP2 antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region and a light chain variable region (VH). In some embodiments, the heavy chain variable region comprises 3 complementary determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises 3 complementary determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0396] In some embodiments, the anti-TROP2 antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody heavy chain constant region. In some embodiments, the anti-TROP2 antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody light chain constant region. In some embodiments, the anti-TROP2 antibodies or antigen-binding fragments thereof of the present invention further comprise a heavy chain constant region and a light chain constant region.

[0397] In some embodiments, the heavy chain variable region of the present invention:

[0398] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 7 and SEQ ID NO: 12; or

[0399] (ii) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 12; or

[0400] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 7 and SEQ ID NO: 12, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0401] In some embodiments, the light chain variable region of the present invention

[0402] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 8 and SEQ ID NO: 14; or

[0403] (ii) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 14; or

[0404] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 8 and SEQ ID NO: 14, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0405] In some embodiments, the three complementarity determining regions (HCDRs) of the present invention, HCDR1, HCDR2, and HCDR3, from the heavy chain variable region are selected from

[0406] (i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 7 or 12;

[0407] (ii) a sequence comprising 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 HCDR regions relative to the sequence of any one of (i),

[0408] wherein said HCDR1-3 are determined according to any CDR scheme, such as Kabat, AbM, Chothia or IMGT or a combination thereof;

[0409] Preferably, the HCDR1 is determined according to the AbM protocol, and the HCDR2 and HCDR3 are each determined according to the Kabat protocol.

[0410] In some embodiments, the three complementarity determining regions (LCDRs) of the present invention, LCDR1, LCDR2, and LCDR3, from the light chain variable region are selected from

[0411] (i) three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 8 or 14, or

[0412] (ii) a sequence comprising 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 LCDR regions relative to the sequence of any one of (i),

[0413] wherein the LCDRs 1 to 3 are determined according to any CDR scheme, such as Kabat, AbM, Chothia or IMGT or a combination thereof;

[0414] Preferably, the LCDRs 1, 2 and 3 are determined according to the Kabat protocol.

[0415] In some embodiments, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, or HCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 1.

[0416] In some embodiments, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, or HCDR2 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 2.

[0417] In some embodiments, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3, or HCDR3 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 3.

[0418] In some embodiments, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 4, or LCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 4.

[0419] In some embodiments, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, or consists of the amino acid sequence, or LCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 5.

[0420] In some embodiments, LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 6, or LCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 6.

[0421] In some embodiments, the heavy chain constant region of the antibody of the present invention is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG4. In some embodiments, the light chain constant region of the antibody of the present invention is the lambda or kappa light chain constant region, preferably the kappa light chain constant region.

[0422] In some embodiments, the heavy chain constant region of an antibody of the invention comprises an Fc region or a mutated Fc region.

[0423] In some embodiments, the Fc region is an IgG Fc from human, e.g., an Fc from human IgG1, an Fc from human IgG2, an Fc from human IgG3, or an Fc from human IgG4. In one embodiment, the Fc region is from human IgG1.

[0424] In some specific embodiments, the anti-TROP2 antibody of the present invention comprises a first heavy chain complementary determining region (HCDR1), a second heavy chain complementary determining region (HCDR2), a third heavy chain complementary determining region (HCDR3), and a first light chain complementary determining region (LCDR1), a second light chain complementary determining region (LCDR2), and a third light chain complementary determining region (LCDR3), wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, and LCDR3 respectively comprise or consist of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; or

[0425] In some embodiments, the VH of the present invention comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, and the HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, or consist of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the LCDR1, LCDR2, and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or consist of the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0426] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises:

[0427] (i) a VH comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 7, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or a VL comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 8, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0428] (ii) a VH comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 12, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or a VL comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 14, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0429] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region and a light chain variable region, wherein

[0430] (i) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 8; or

[0431] (ii) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 12, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 14.

[0432] In some embodiments, the anti-TROP2 antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody heavy chain. In some embodiments, the anti-TROP2 antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody light chain. In some embodiments, the anti-TROP2 antibodies or antigen-binding fragments thereof of the present invention further comprise a heavy chain and a light chain.

[0433] In some embodiments, the heavy chain comprises

[0434] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 9 or 15;

[0435] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 9 or 15; or

[0436] (iii) comprises an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 9 or 15, or consists of said amino acid sequence.

[0437] In some embodiments, the light chain comprises

[0438] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 10 or 16;

[0439] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 10 or 16; or

[0440] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 10 or 16.

[0441] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO:9, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO:10.

[0442] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 15, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO: 16.

[0443] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise or consist of two said heavy chains and two said light chains, optionally, the two said heavy chains are identical and / or the two said light chains are identical.

[0444] In one embodiment of the present invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0445] In some embodiments, the anti-TROP2 antibodies or antigen-binding fragments thereof of the present invention have one or more of the following characteristics:

[0446] (i) exhibiting the same or similar binding affinity and / or specificity to TROP2 as the antibodies of the present invention;

[0447] (ii) inhibiting (e.g., competitively inhibiting) the binding of the antibody of the present invention to TROP2;

[0448] (iii) binds to the same or overlapping epitope as an antibody of the invention;

[0449] (iv) competing with the antibodies of the present invention for binding to TROP2;

[0450] (v) possess one or more biological properties of an antibody of the invention.

[0451] In some embodiments, the anti-TROP2 antibody of the present invention is an antibody in the form of IgG1, an antibody in the form of IgG2, an antibody in the form of IgG3, or an antibody in the form of IgG4, preferably, an antibody in the form of IgG1.

[0452] In some embodiments, the anti-TROP2 antibody is a monoclonal antibody.

[0453] In some embodiments, the anti-TROP2 antibody is humanized.

[0454] In some embodiments, the anti-TROP2 antibody is a chimeric antibody.

[0455] In one embodiment, the anti-TROP2 antibodies of the present invention also encompass antibody fragments thereof (e.g., antigen-binding fragments), preferably antibody fragments selected from the following: Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies such as VHH, dAb (domain antibody), bivalent antibodies or linear antibodies.

[0456] In one embodiment, the anti-TROP2 antibody is a full-length antibody.

[0457] In one embodiment, the anti-TROP2 antibody also encompasses multispecific antibodies such as bispecific antibodies.

[0458] In some embodiments, the anti-FRα antibodies or antigen-binding fragments thereof of the present invention comprise three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the anti-FRα antibodies or antigen-binding fragments thereof of the present invention comprise three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, the anti-FRα antibodies or antigen-binding fragments thereof of the present invention comprise three complementarity determining regions (HCDRs) from the heavy chain variable region and three complementarity determining regions (LCDRs) from the light chain variable region.

[0459] In some aspects, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH). In some aspects, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region (VL). In some aspects, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain variable region comprises three complementary determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementary determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0460] In some embodiments, the anti-FRα antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody heavy chain constant region. In some embodiments, the anti-FRα antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody light chain constant region. In some embodiments, the anti-FRα antibodies or antigen-binding fragments thereof of the present invention further comprise a heavy chain constant region and a light chain constant region.

[0461] In some embodiments, the heavy chain variable region of the present invention:

[0462] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 33; or

[0463] (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 33; or

[0464] (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any one of (i) to (ii) or consists of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR region.

[0465] In some embodiments, the light chain variable region of the present invention

[0466] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 34; or

[0467] (ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 34;

[0468] or

[0469] (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from any one of (i) to (ii), or consists of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR region.

[0470] In some embodiments, the three complementarity determining regions (HCDRs) of the present invention, HCDR1, HCDR2, and HCDR3, from the heavy chain variable region are selected from

[0471] (i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 33; or

[0472] (i) a sequence comprising 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 HCDR regions relative to the sequence of any one of (i),

[0473] wherein the HCDRs can be determined according to any scheme for determining CDRs, for example, according to the schemes of Kabat, AbM, Chothia, Contact or IMGT or a combination thereof;

[0474] For example, the HCDR1 is determined according to a union of the Kabat and Chothia schemes, and the HCDR2 and HCDR3 are each determined according to the Kabat scheme.

[0475] In some embodiments, the three complementarity determining regions (LCDRs) of the present invention, LCDR1, LCDR2, and LCDR3, from the light chain variable region are selected from

[0476] (i) three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 34, or

[0477] (i) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three LCDR regions relative to the sequence of any one of (i),

[0478] wherein the LCDR can be determined according to any scheme for determining CDRs, for example, according to the schemes of Kabat, AbM, Chothia, Contact or IMGT, or a combination thereof;

[0479] For example, the LCDR1, LCDR2 and LCDR3 are each determined according to the Kabat protocol.

[0480] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the invention comprises:

[0481] The three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO: 33, and the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO: 34;

[0482] wherein the HCDR and LCDR can be determined according to any scheme for determining CDRs, for example, according to the schemes of Kabat, AbM, Chothia, Contact or IMGT or a combination thereof;

[0483] For example, the HCDR1 is determined according to a union of the Kabat and Chothia schemes, the HCDR2 and HCDR3 are each determined according to the Kabat scheme, and the LCDR1, LCDR2, and LCDR3 are each determined according to the Kabat scheme.

[0484] In some embodiments, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 27, or HCDR1 comprises an amino acid sequence having one, two or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 27.

[0485] In some embodiments, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 28, or HCDR2 comprises an amino acid sequence having one, two or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 28.

[0486] In some embodiments, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 29, or HCDR3 comprises an amino acid sequence having one, two or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 29.

[0487] In some embodiments, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 30, or LCDR1 comprises an amino acid sequence having one, two or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 30.

[0488] In some embodiments, LCDR2 comprises the amino acid sequence of SEQ ID NO: 31, or consists of the amino acid sequence, or LCDR2 comprises an amino acid sequence having one, two or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 31.

[0489] In some embodiments, LCDR3 comprises the amino acid sequence of SEQ ID NO: 32, or consists of the amino acid sequence, or LCDR3 comprises an amino acid sequence having one, two or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 32.

[0490] In some specific embodiments, the anti-FRα antibody of the invention comprises a first heavy chain complementarity determining region (HCDR1), a second heavy chain complementarity determining region (HCDR2), a third heavy chain complementarity determining region (HCDR3), and a first light chain complementarity determining region (LCDR1), a second light chain complementarity determining region (LCDR2), and a third light chain complementarity determining region (LCDR3), wherein

[0491] The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively contain the amino acid sequences shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or respectively consist of the amino acid sequences shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32.

[0492] In some embodiments, the VH of the invention comprises HCDR1, HCDR2, HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein;

[0493] The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively contain the amino acid sequences shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or respectively consist of the amino acid sequences shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32.

[0494] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises:

[0495] A VH comprising the amino acid sequence shown in SEQ ID NO: 33, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consisting of said amino acid sequence, and / or a VL comprising the amino acid sequence shown in SEQ ID NO: 34, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consisting of said amino acid sequence.

[0496] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 33, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 34.

[0497] In one embodiment of the invention, the amino acid changes described herein include amino acid replacements, insertions, or deletions. In some embodiments, the amino acid changes described herein are conservative amino acid changes. Preferably, the amino acid changes described herein are amino acid replacements, preferably conservative replacements. In a preferred embodiment, the amino acid changes described herein occur in regions outside the CDRs (e.g., in the FRs). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region.

[0498] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention further comprises an antibody heavy chain. In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention further comprises an antibody light chain. In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain and a light chain. In some embodiments, the antibody heavy chain of the present invention comprises a heavy chain variable region and a heavy chain constant region, or consists of a heavy chain variable region and a heavy chain constant region. In some embodiments, the antibody light chain of the present invention comprises a light chain variable region and a light chain constant region, or consists of a light chain variable region and a light chain constant region. In some embodiments, the antibody of the present invention comprises two heavy chains and two light chains, or consists of two heavy chains and two light chains. In some embodiments, the antibody of the present invention comprises two identical heavy chains and two identical light chains, or consists of them.

[0499] In some embodiments, the heavy chain constant region of an anti-FRα antibody of the present invention is an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as a human IgG1, IgG2, IgG3, or IgG4 constant region. In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region is an IgG1 heavy chain constant region, such as an amino acid sequence as set forth in SEQ ID NO: 46, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0500] In some embodiments, the anti-FRα antibody light chain constant region of the present invention is a lambda or kappa light chain constant region, preferably a kappa light chain constant region, such as a human lambda or kappa light chain constant region. In some embodiments, the light chain constant region is a (human) lambda light chain constant region. In some embodiments, the lambda light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 38 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 38 and does not comprise a cysteine ​​mutation. In some embodiments, the light chain constant region is a (human) kappa light chain constant region. In some embodiments, the kappa light chain constant region comprises the amino acid sequence set forth in SEQ ID NO:45, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:45.

[0501] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention is an antibody or antigen-binding fragment thereof having a cysteine ​​mutation in the light chain constant region, wherein the antibody or antigen-binding fragment thereof comprises one or two Lambda light chain constant regions and has a cysteine ​​substitution at position 160 (EU numbering) of the Lambda light chain constant region (LLC160C or LLC160), and / or has a cysteine ​​substitution at position 166 (EU numbering) of the Lambda light chain constant region (LLC166C or LLC166).

[0502] In some embodiments, the light chain constant region of the anti-FRα antibody or antigen-binding fragment thereof of the present invention has a cysteine ​​mutation at position 160 (Eu numbering) relative to the wild-type Lambda light chain constant region, such as the amino acid sequence shown in SEQ ID NO: 38.

[0503] In some embodiments, the Lambda light chain constant region with a cysteine ​​mutation at position 160 comprises the amino acid sequence set forth in SEQ ID NO:39, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:39 and comprises the amino acid sequence VKAGVCTTTPS (SEQ ID NO:42).

[0504] In some embodiments, the light chain constant region of the anti-FRα antibody or antigen-binding fragment thereof of the present invention has a cysteine ​​mutation at position 166 (Eu numbering) relative to the wild-type Lambda light chain constant region, such as the amino acid sequence shown in SEQ ID NO: 38.

[0505] In some embodiments, the Lambda light chain constant region with a cysteine ​​mutation at position 166 comprises the amino acid sequence set forth in SEQ ID NO:40, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:40 and comprises the amino acid sequence TTTPSCQSNNK (SEQ ID NO:43).

[0506] In some embodiments, the light chain constant region of the anti-FRα antibody or antigen-binding fragment thereof of the present invention has cysteine ​​mutations at positions 160 and 166 (Eu numbering) relative to the wild-type Lambda light chain constant region, such as the amino acid sequence shown in SEQ ID NO: 38.

[0507] In some embodiments, the Lambda light chain constant region having cysteine ​​mutations at positions 160 and 166 comprises the amino acid sequence set forth in SEQ ID NO:41, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:41 and comprises the amino acid sequence VKAGVCTTTPSCQSNNK (SEQ ID NO:44).

[0508] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise

[0509] (i) one or both Lambda light chain constant regions having a cysteine ​​mutation at position 160 (Eu numbering); and / or

[0510] (ii) one or both Lambda light chain constant regions with a cysteine ​​mutation at position 166 (Eu numbering).

[0511] The anti-FRα antibody or antigen-binding fragment thereof of the present invention can be any form of antibody or antigen-binding fragment thereof known in the art, such as monoclonal, chimeric, humanized, fully human, bispecific, or multispecific antibody or antibody fragment thereof.

[0512] In some embodiments, the heavy chain of an anti-FRα antibody of the invention

[0513] comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 35;

[0514] comprising or consisting of an amino acid sequence selected from SEQ ID NO: 35; or

[0515] An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 35.

[0516] In some embodiments, the light chain of an anti-FRα antibody of the invention

[0517] comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 36 or 37;

[0518] comprising or consisting of an amino acid sequence selected from SEQ ID NO: 36 or 37; or

[0519] An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 36 or 37.

[0520] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises two identical heavy chains and two identical light chains, wherein

[0521] The heavy chain comprises a VH as described herein, or a HCDR1, HCDR2, and HCDR3 as described herein, and an Fc region and CH1 as described herein, or a heavy chain constant region as described herein; and / or

[0522] The light chain comprises a VL described herein or LCDR1, LCDR2, and LCDR3 described herein, and a kappa light chain constant region described herein.

[0523] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises two identical heavy chains and two identical light chains, wherein

[0524] The heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 35, and / or

[0525] The light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:36.

[0526] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises two identical heavy chains and two identical light chains, wherein

[0527] The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 35, and / or

[0528] The light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 36.

[0529] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises two identical heavy chains and two identical light chains, wherein

[0530] The heavy chain comprises a VH as described herein, or a HCDR1, HCDR2, and HCDR3 as described herein, and an Fc region and CH1 as described herein, or a heavy chain constant region as described herein;

[0531] The light chain comprises a VL as described herein or LCDR1, LCDR2, and LCDR3 as described herein, and a lambda light chain constant region comprising LLC160C and / or 166C as described herein.

[0532] In a specific embodiment, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises two identical heavy chains and two identical light chains, wherein

[0533] The heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 35, and / or

[0534] The light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:37.

[0535] In a more specific embodiment, the anti-FRα antibody or antigen-binding fragment thereof of the invention comprises two identical heavy chains and two identical light chains, wherein

[0536] The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 35, and / or

[0537] The light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 37.

[0538] In some embodiments, the anti-FRα antibodies or antigen-binding fragments thereof of the present invention are chimeric or humanized antibodies.

[0539] In some embodiments, the antigen-binding fragment of an anti-FRα antibody of the invention is, for example, Fv, Fab, Fab', Fab'-SH, F(ab')2; dAb (domain antibody); a linear antibody; a single-chain antibody (e.g., scFv); a single-domain antibody such as VHH; a diabody or fragment thereof; or a camelid antibody.

[0540] In one embodiment, the anti-FRα antibody is a full-length antibody.

[0541] In one embodiment, the anti-FRα antibody also encompasses multispecific antibodies such as bispecific antibodies.

[0542] In some embodiments, the anti-FRα antibodies or antigen-binding fragments thereof of the present invention have good endocytic activity on FRα-positive cells, such as FRα-positive tumor cells.

[0543] In some embodiments, the present invention also provides a fusion protein comprising the anti-FRα antibody or antigen-binding fragment thereof of the present invention.

[0544] In some embodiments, the present invention also provides an immunoconjugate comprising an anti-FRα antibody or antigen-binding fragment thereof of the present invention, and further comprising a payload coupled to the antibody or antigen-binding fragment thereof. In some embodiments, the payload is a toxin, a small molecule drug / agent, a cytotoxic agent, an apoptotic agent, a chelating agent, an immunomodulator / immunostimulator / immunoagonist, an oligonucleotide, a polypeptide, a peptide epitope, a radionuclide, a prodrug, and the like. In some preferred embodiments, the payload is an immunostimulator / immunoactivator. In some more preferred embodiments, the payload is a TLR agonist. In some more preferred embodiments, the payload is a TLR7 / 8 agonist.

[0545] Therefore, the present invention provides antibodies or antigen-binding fragments thereof targeting FRα and antibody-immunoagonist conjugates (ISACs) targeting FRα.

[0546] In one aspect, the antibodies or antigen-binding fragments thereof of the present invention are subjected to cysteine ​​modification, such that one or more amino acids on the heavy or light chain of the antibodies or antigen-binding fragments thereof are mutated to cysteine. The sulfhydryl groups on the cysteine ​​can undergo nucleophilic reactions with small molecules (e.g., immune agonists, particularly TLR agonists, more particularly TLR7 / 8 agonists) having maleimide linkers, thereby coupling the small molecules to the cysteine ​​residues to prepare site-specifically coupled ISAC molecules.

[0547] In some embodiments, the Ab in the ADC of the present invention (including those of formula (II) or (III)) comprises cysteine. In some embodiments, in the ADC of the present invention, the Ab is linked to a linker via its cysteine ​​(e.g., natural and / or cysteine ​​mutation-introduced), for example, the linker is coupled to the Ab via the thiol group of cysteine.

[0548] In some embodiments, the Ab is coupled to the linker via a native cysteine, eg, the linker is coupled to the Ab via the sulfhydryl group of the cysteine ​​with the disulfide bond opened (eg, random coupling).

[0549] In some embodiments, the Ab is coupled to the linker via a cysteine ​​introduced by a cysteine ​​mutation thereof, for example, the Ab is coupled to the linker via a cysteine ​​residue that is not paired or otherwise part of an intramolecular or intermolecular disulfide bond (e.g., a cysteine ​​obtained after a cysteine ​​mutation) (e.g., site-directed coupling), or, for example, the linker is coupled to the Ab via the sulfhydryl group of a cysteine ​​whose disulfide bond is opened (e.g., random coupling or site-directed coupling).

[0550] In some embodiments, in the ADC molecules of the present invention, the Ab is conjugated to one toxin via a linker through a cysteine ​​introduced by a cysteine ​​mutation thereof (e.g., site-specific conjugation), and is conjugated to another toxin through its native cysteine ​​(e.g., random conjugation).

[0551] In some embodiments, the Ab in the ADC molecule of the present invention comprises a cysteine ​​introduced by a cysteine ​​mutation, such that the linker is coupled to the antibody via the sulfhydryl group of the mutated cysteine. In some embodiments, the Ab comprises one or more non-cysteine ​​mutations to cysteine ​​on its heavy or light chain, such that the linker is coupled to the cysteine, for example, site-directed coupling. Antibodies or antigen-binding fragments thereof comprising the cysteine ​​mutations suitable for use in the ADC of the present invention are described in detail herein, wherein the cysteine ​​obtained after the mutation is coupled to the linker, for example, site-directed coupling.

[0552] In some embodiments, in the ISAC molecules of the invention, the Ab is conjugated to the linker via a cysteine ​​obtained by mutation as follows:

[0553] (i) a cysteine ​​obtained by mutation of cysteine ​​at position 160 in one or both Lambda light chain constant regions; and / or

[0554] (ii) a cysteine ​​obtained by mutation of cysteine ​​at position 166 in one or both Lambda light chain constant regions.

[0555] Any cysteine ​​residue obtained after any cysteine ​​residue mutation of any antibody or antigen-binding fragment thereof mentioned herein can be used for coupling with a linker, such as site-directed coupling, to obtain the immunostimulatory molecule of the present invention.

[0556] In one aspect, the antibodies or antigen-binding fragments thereof of the present invention have a cysteine ​​mutation at a relatively hidden position in the constant region, thereby conferring improved stability and / or hydrophilicity to the immunoconjugate (e.g., ISAC) comprising the same. As defined herein, a "cysteine ​​mutation" refers to the substitution of an amino acid in a protein that is not originally cysteine ​​with cysteine.

[0557] In one aspect, the present invention provides nucleic acids encoding any chain or any monomer or domain of an antibody or antigen-binding fragment thereof of the present invention. Polynucleotide sequences encoding each chain can be generated using methods well known in the art. For example, when expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid can exhibit human TROP2 antigen binding ability. For example, in some embodiments, the nucleic acid encoding the variable region of the heavy chain and / or light chain is operably linked in frame with the nucleic acid encoding the constant region of the heavy chain and / or light chain, thereby generating nucleic acids encoding the antibody heavy chain and / or light chain when expressed from a suitable expression vector.

[0558] In one aspect, the present invention provides nucleic acids encoding any of the anti-TROP2 antibodies or fragments thereof described herein. The nucleic acids may comprise nucleic acids encoding the amino acid sequences of the light chain variable region and / or heavy chain variable region of the antibody, or nucleic acids encoding the amino acid sequences of the light chain and / or heavy chain of the antibody.

[0559] For example, the nucleic acids of the present invention include nucleic acids encoding an amino acid sequence selected from any one of SEQ ID NOs: 1-10, 12, and 14-16, or nucleic acids encoding 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 any one of SEQ ID NOs: 1-10, 12, and 14-16. 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. Nucleic acid sequences encoding the molecules of the present invention can be generated using methods well known in the art, such as de novo solid-phase DNA synthesis or PCR amplification. To facilitate production and purification, a secretory signal peptide and / or a tag peptide that facilitates purification can be fused to the N-terminus of the heavy and / or light chains of the antibody.

[0560] In another aspect, the present invention provides a nucleic acid encoding any of the anti-FRα antibodies or fragments thereof described herein. The nucleic acid may comprise a nucleic acid encoding an amino acid sequence of the light chain variable region and / or heavy chain variable region of the antibody, or a nucleic acid encoding an amino acid sequence of the light chain and / or heavy chain of the antibody.

[0561] For example, the nucleic acids of the present invention include nucleic acids encoding an amino acid sequence selected from any one of SEQ ID NOs: 27-46, or nucleic acids encoding 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 any one of SEQ ID NOs: 27-46. 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. Nucleic acid sequences encoding the molecules of the present invention can be generated using methods well known in the art, such as de novo solid-phase DNA synthesis or PCR amplification. To facilitate production and purification, a secretory signal peptide and / or a tag peptide that facilitates purification can be fused to the N-terminus of the heavy and / or light chains of the antibody.

[0562] The polynucleotide encoding the polypeptide chain of the 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 expression vectors. Once an expression vector comprising one or more nucleic acid molecules of the present invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection or other conventional techniques.

[0563] The present invention also provides vectors comprising nucleic acids of the present invention. 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, lambda phages, or yeast artificial chromosomes (YACs). In preferred embodiments, the expression vector of the present invention is a pcDNA vector, such as a pcDNA3.1 expression vector. In other preferred embodiments, the expression vector of the present invention is a pTT5 vector or an expression vector derived from pTT5.

[0564] In one embodiment, a host cell comprising the vector is provided. The present invention also provides a host cell comprising the nucleic acid or the vector. Host cells suitable for replication and support expression of the antibodies of the present invention are well known in the art. Such cells can be transfected or transduced with specific expression vectors, and large quantities of vector-containing cells can be grown for inoculating large-scale fermenters, thereby obtaining sufficient amounts of antibodies for clinical applications. Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. After expression, the antibodies can be separated from the bacterial cell paste in the soluble fraction and can be further purified.

[0565] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells (e.g., CHO-S or CHO-K) or 293 cells (e.g., 293F or HEK293 cells)) or other cells suitable for preparing antibodies or fragments thereof. In one embodiment, the host cell is prokaryotic, for example, a bacterium, such as Escherichia coli.

[0566] For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. For example, fungal and yeast strains whose glycosylation pathways have been "humanized" result in the production of antibodies with partially or fully human glycosylation patterns. 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 modified to be suitable for suspension growth can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 lines (COS-7) transformed with SV40; 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.

[0567] III. Immunoconjugates

[0568] The present invention provides an antibody-immunoagonist conjugate having formula (I):

[0569] Ab-(LD) p (I)

[0570] or a pharmaceutically acceptable salt or solvate thereof,

[0571] in:

[0572] Ab is an antibody or a fragment thereof that binds to TROP2 (eg, human TROP2);

[0573] L is a linker;

[0574] D is a drug, preferably an anti-tumor compound; and

[0575] p is 1 to 16, such as 1-10, 1-9, 2-8, 4-10, 3-7, 4-6, 2-6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12.

[0576] In some embodiments, Ab in formula (I) of the present invention is the aforementioned anti-TROP2 antibody or antigen-binding fragment thereof or anti-FRα antibody or antigen-binding fragment thereof of the present invention.

[0577] In some embodiments, D in formula (I) of the present invention can be any TLR7 / 8 agonist compound, such as the compounds of formula (D-1) to (D-5) defined above, such as the compound of formula (D-1'), such as a compound selected from the following:

[0578] IV. Preparation of Antibodies and ISAC Molecules of the Invention

[0579] The present invention provides methods for preparing antibodies, for example, preparing the anti-TROP2 antibody or antigen-binding fragment thereof or the anti-FRα antibody or antigen-binding fragment thereof of the present invention, and preparing Abs for use in the ISAC of the present invention.

[0580] In one embodiment, the method comprises culturing a host cell comprising a nucleic acid encoding the 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 expression of the antibody or its peptide chains, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0581] Abs prepared as described herein can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these will be apparent to those skilled in the art.

[0582] In some embodiments, the present invention relates to a method for preparing an anti-TROP2 or FRα antibody or an antigen-binding fragment thereof, the method comprising:

[0583] a) culturing the host cell of the present invention under conditions suitable for expressing a nucleic acid encoding an anti-TROP2 or FRα antibody or antigen-binding fragment thereof of the present invention,

[0584] b) optionally isolating said antibody or antigen-binding fragment thereof,

[0585] c) Optionally, the method further comprises recovering the anti-TROP2 or FRα antibody or antigen-binding fragment thereof from the host cell. Optionally, the antibody is purified, for example, by Protein A purification.

[0586] Another aspect of the present invention provides a method for preparing an ISAC using the antibody of the present invention. "ISAC" herein is defined as an antibody coupled to a biologically and / or pharmaceutically active active substance (D) via a linker (L). The method comprises coupling an antibody of the present invention (Ab) to one or more active substances D via one or more linkers (L) as defined herein. Preferably, the linker-active substance is site-specifically coupled to the antibody.

[0587] In some embodiments, the method comprises the steps of:

[0588] (a) adding the antibody Ab to a buffer solution, adding a reducing agent, and then incubating;

[0589] (b) adding a linker-payload to the reaction solution in step (a) for coupling to obtain a crude product; and

[0590] (c) optionally purifying the crude product to obtain the antibody drug conjugate of the present invention;

[0591] wherein Ab is as defined above.

[0592] It will be understood that the linker-payload reacts with Ab to provide the -LD moiety in the compound of formula (I), and where -LD is clearly defined, the structure of the linker-payload can be determined.

[0593] In some embodiments, the buffer solution in step a) is a PBS buffer, preferably, having a pH of 5.0-9.0, such as 6.0-8.0.

[0594] In some embodiments, the reducing agent of step a) is TCEP.

[0595] In some embodiments, the linker-payload has the structure shown in Formula (II), L-(D) m (II)

[0596] wherein D and L are as defined above.

[0597] In some embodiments, the steps are performed under the specific reaction conditions disclosed in the Examples.

[0598] It should be noted that embodiments in which the ranges or specific values ​​of the specific reaction conditions disclosed in the examples are varied by 100%, 80%, 60%, 40%, 20% or 10% are also contemplated by the present invention.

[0599] V. Pharmaceutical Compositions

[0600] In some embodiments, the present invention provides a composition comprising any of the antibodies or fragments thereof, ISAC molecules, or pharmaceutically acceptable salts thereof described herein, preferably a pharmaceutical composition or pharmaceutical formulation. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, e.g., a pharmaceutical composition, comprises an antibody or fragment thereof, ISAC molecule of the present invention in combination with one or more other therapeutic agents.

[0601] The present invention also includes compositions (including pharmaceutical compositions) comprising the antibodies or fragments thereof, ISAC molecules, or pharmaceutically acceptable salts thereof of the present invention. These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

[0602] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0603] For the use of pharmaceutical excipients and their applications, see also "Handbook of Pharmaceutical Excipients", 8th edition, RC Rowe, PJ Eskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0604] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0605] Medicaments comprising the ISACs described herein can be prepared by mixing an antibody or fragment thereof or ISAC molecule of the invention having the desired degree of purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.

[0606] The pharmaceutical composition or preparation of the present invention may also include more than one active ingredient, which is required for the specific indication being treated, preferably having those active ingredients of complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, including chemotherapeutics, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (such as immune checkpoint inhibitors or agonists). The active ingredient is suitably present in combination in an amount effective for the purpose.

[0607] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films, or microcapsules.

[0608] VI. Pharmaceutical Combinations and Kits

[0609] In some embodiments, the present invention also provides a drug combination or drug combination product comprising an antibody or fragment thereof or ISAC molecule of the present invention, and one or more other therapeutic agents (e.g., therapeutic agents including chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (e.g., immune checkpoint inhibitors or agonists), etc.).

[0610] In some embodiments, the other therapeutic agent is an antibody-drug conjugate targeting TROP2, a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0611] In some embodiments, the other therapeutic agent is an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate disclosed in PCT / CN2024 / 121813.

[0612] In some embodiments, the additional therapeutic agent is an antibody-drug conjugate of the formula, a stereoisomer, or a pharmaceutically acceptable salt or solvate thereof,

[0613] Wherein A represents an antibody or fragment thereof targeting TROP2:

[0614] p' is a value between 1-15, for example, 2-10, 2-8, 2-6, 2-5, 3-5, 3.5-4.5.

[0615] In some embodiments, the antibody targeting TROP2 or its fragment (e.g., antigen-binding fragment) suitable for constructing the above-mentioned antibody-drug conjugate may be a mammalian-derived (e.g., human or mouse), humanized or chimeric antibody or antibody fragment targeting TROP2. In some embodiments, the antibody targeting TROP2 or its fragment suitable for constructing the above-mentioned antibody-drug conjugate is an anti-TROP2 antibody or its fragment, such as an antigen-binding fragment, as defined above.

[0616] In some embodiments, the TROP2-targeting antibody or fragment thereof suitable for constructing the above-mentioned antibody-drug conjugate comprises a first heavy chain complementarity determining region (HCDR1), a second heavy chain complementarity determining region (HCDR2), a third heavy chain complementarity determining region (HCDR3), and a first light chain complementarity determining region (LCDR1), a second light chain complementarity determining region (LCDR2), and a third light chain complementarity determining region (LCDR3), wherein:

[0617] The HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 17;

[0618] The HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 18;

[0619] The HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 19;

[0620] The LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 20;

[0621] The LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 21;

[0622] The LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 22.

[0623] In some embodiments, the TROP2-targeting antibody or fragment thereof suitable for constructing the above-mentioned antibody-drug conjugate comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, or consist of the amino acid sequences shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively.

[0624] In some embodiments, the TROP2-targeted antibody or fragment thereof suitable for constructing the above-mentioned antibody-drug conjugate comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23 or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 23, or consists of the amino acid sequence, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 24 or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 24, or consists of the amino acid sequence.

[0625] In some embodiments, the TROP2-targeted antibody or fragment thereof suitable for constructing the above-mentioned antibody-drug conjugate comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23 and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 24. In some embodiments, the TROP2-targeted antibody or fragment thereof suitable for constructing the above-mentioned antibody-drug conjugate comprises a heavy chain variable region with a sequence of SEQ ID NO: 23 and a light chain variable region with a sequence of SEQ ID NO: 24.

[0626] In some embodiments, the TROP2-targeted antibody or fragment thereof suitable for constructing the above-mentioned antibody-drug conjugate comprises a heavy chain and a light chain, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 25 or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 25, or consists of the amino acid sequence, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 26 or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 26, or consists of the amino acid sequence.

[0627] In some embodiments, the TROP2-targeted antibody or fragment thereof suitable for constructing the above-mentioned antibody-drug conjugate comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 25 and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 26. In some embodiments, the TROP2-targeted antibody or fragment thereof suitable for constructing the above-mentioned antibody-drug conjugate comprises a heavy chain with a sequence of SEQ ID NO: 25 and a light chain with a sequence of SEQ ID NO: 24.

[0628] In some specific embodiments, the antibody targeting TROP2 suitable for constructing the above-mentioned antibody-drug conjugate is a full-length antibody. In a specific embodiment, the antibody targeting TROP2 comprises two heavy chains as defined above and two light chains as defined above, or consists of two of the heavy chains and two of the light chains.

[0629] In some specific embodiments, the antibody targeting TROP2 or its fragment suitable for constructing the above-mentioned antibody-drug conjugate is a multispecific antibody such as a bispecific antibody. In some specific embodiments, the antibody targeting TROP2 suitable for constructing the above-mentioned antibody-drug conjugate is a monoclonal antibody. In some specific embodiments, the antibody targeting TROP2 suitable for constructing the above-mentioned antibody-drug conjugate is a chimeric antibody or a humanized antibody. In some specific embodiments, the antibody fragment targeting TROP2 suitable for constructing the above-mentioned antibody-drug conjugate is selected from Fab, Fab', Fab'-SH, Fv, single-chain antibody (such as scFv), (Fab')2, single domain antibody such as VHH, dAb (domain antibody), bivalent antibody or linear antibody.

[0630] In some embodiments, the antibody or fragment thereof targeting TROP2 is an antibody or fragment thereof targeting TROP2 in PCT / CN2024 / 121813, such as hRS7 antibody. Patent application PCT / CN2024 / 121813 is incorporated herein in its entirety.

[0631] In some embodiments, the other therapeutic agent is an antibody-drug conjugate targeting FRα, a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof. In some specific embodiments, the antibody or fragment thereof targeting FRα is Mirvetuximab from ImmunoGen, disclosed in U.S. Patent Application Publication No. US20200362029A1. In some specific embodiments, the antibody or fragment thereof targeting FRα is FR57 from ImmunoGen, disclosed in U.S. Patent Application Publication No. US20200362029A1. In some specific embodiments, the antibody-drug conjugate targeting FRα is In some specific embodiments, the targeting FRα antibody-drug conjugate is FR57-Dxd. Patent application US20200362029A1 is incorporated herein in its entirety.

[0632] The above-mentioned antibody-drug conjugates can be prepared according to the methods described in the following examples or the methods described in the related applications PCT / CN2024 / 121813 and US20200362029A1.

[0633] Another object of the present invention is to provide a kit comprising the pharmaceutical combination of the present invention, preferably in the form of a pharmaceutical dosage unit, whereby dosage units can be provided according to a dosing regimen or a drug administration interval.

[0634] In one embodiment, the kit of parts of the present invention comprises in the same package:

[0635] - a first container containing a pharmaceutical composition comprising an antibody or fragment thereof, and / or ISAC molecule of the present invention;

[0636] - A second container containing a pharmaceutical composition comprising an additional therapeutic agent.

[0637] VII. Uses and Methods

[0638] In one aspect, the present invention provides a method for preventing or treating a tumor (eg, cancer) in a subject, comprising administering to the subject an effective amount of an antibody and / or ISAC molecule, pharmaceutical composition, pharmaceutical combination, or kit of the present invention.

[0639] In some embodiments, the tumor, such as cancer, includes solid tumors and blood tumors and metastatic lesions. In one embodiment, the example of a solid tumor includes a malignant tumor. The cancer can be in the early, middle or late stages or be a metastatic cancer.

[0640] In some embodiments, the tumor, such as cancer, is TROP2 positive, for example, it comprises tumor cells expressing TROP2. In some embodiments, the patient's tumor comprises tumor cells expressing TROP2. In some embodiments, the patient's tumor cells express TROP2, such as moderate expression TROP2, preferably high expression TROP2. In some embodiments, the tumor (e.g., cancer) patient's tumor tissue has (e.g., elevated levels of, such as nucleic acid or protein levels of) TROP2, for example, compared with the same tissue of a healthy individual or with the TROP2 levels in a healthy tissue adjacent to the patient's tumor tissue. In some embodiments, the patient's tumor cells have (e.g., elevated levels of, such as nucleic acid or protein levels of) TROP2, for example, compared with the TROP2 levels in the same cell of a healthy individual or with the TROP2 levels in a healthy cell adjacent to the patient's tumor cell.

[0641] In a specific embodiment, the antibodies and / or ISAC molecules of the present invention are capable of killing tumor cells and / or inhibiting the proliferation of tumor cells, such as tumor cells expressing TROP2, such as lung cancer cells, head and neck tumor cells, prostate cancer cells, melanoma cells, pancreatic cancer cells, breast cancer cells, esophageal cancer cells, cervical cancer cells, renal cancer cells, bladder cancer cells or ovarian cancer cells.

[0642] In some embodiments, the tumor, such as cancer, is FRα-positive, for example, it comprises tumor cells expressing FRα. In some embodiments, the patient's tumor comprises tumor cells expressing FRα. In some embodiments, the patient's tumor cells express FRα, for example, moderate expression of FRα, preferably high expression of FRα. In some embodiments, the tumor (e.g., cancer) patient's tumor tissue has (e.g., elevated levels, such as nucleic acid or protein levels) FRα, for example, compared to the FRα levels in the same tissue of a healthy individual or in a healthy tissue adjacent to the patient's tumor tissue. In some embodiments, the patient's tumor cells have (e.g., elevated levels, such as nucleic acid or protein levels), for example, compared to the FRα levels in the same cells of a healthy individual or in healthy cells adjacent to the patient's tumor cells.

[0643] In a specific embodiment, the antibodies and / or ISAC molecules of the present invention are capable of killing tumor cells and / or inhibiting the proliferation of tumor cells, such as tumor cells expressing FRα, such as lung cancer cells, head and neck tumor cells, prostate cancer cells, melanoma cells, pancreatic cancer cells, breast cancer cells, esophageal cancer cells, cervical cancer cells, renal cancer cells, bladder cancer cells or ovarian cancer cells.

[0644] In some embodiments, the tumor is an immune evasion tumor.

[0645] In some embodiments, the tumor is a cancer, such as lung cancer, head and neck cancer, prostate cancer, melanoma, pancreatic cancer, breast cancer, esophageal cancer, cervical cancer, kidney cancer, bladder cancer, or ovarian cancer.

[0646] The subject can be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., an individual suffering from a disease described herein or at risk of suffering from a disease described herein). In one embodiment, the subject suffers from a disease described herein (e.g., cancer) or is at risk of suffering from a disease described herein. In some embodiments, the subject receives or has received other treatments, e.g., chemotherapy and / or radiotherapy. In some embodiments, the subject has previously received or is receiving immunotherapy.

[0647] In other aspects, the present invention provides the use of the above-mentioned antibodies and / or ISAC molecules or pharmaceutical compositions or pharmaceutical combinations or kits in the production or preparation of medicaments for the uses described herein, such as for preventing or treating the relevant diseases or conditions mentioned herein.

[0648] In some embodiments, the antibodies and / or ISAC molecules or pharmaceutical compositions or pharmaceutical combinations or kits of the invention delay the onset of a disorder and / or symptoms associated with a disorder.

[0649] In some embodiments, the antibodies and / or ISAC molecules or pharmaceutical compositions of the invention can also be administered in combination with one or more other therapies, e.g., treatment modalities and / or other therapeutic agents, for the uses described herein, e.g., for preventing and / or treating the relevant diseases or conditions mentioned herein.

[0650] In some embodiments, the treatment modality includes surgery; radiation therapy, localized or focused irradiation, and the like.

[0651] In some embodiments, the therapeutic agent is selected from a chemotherapeutic agent, an angiogenesis inhibitor, a cytokine, a cytotoxic agent, other antibodies, a small molecule drug, or an immunomodulator (e.g., an immune checkpoint inhibitor or agonist).

[0652] Exemplary additional antibodies include antibodies that specifically bind to immune checkpoints.

[0653] Combination therapies of the invention encompass combined administration (e.g., two or more therapeutic agents contained in the same formulation or separate formulations), and separate administration, in which case administration of the antibodies and / or ISAC molecules of the invention can occur prior to, concurrently with, and / or after administration of the other therapeutic agents and / or agents.

[0654] The route of administration of the pharmaceutical composition is according to known methods, for example, orally, by intravenous injection, intraperitoneally, intracerebral (intraparenchymal), intracerebroventricular, intramuscularly, intraocularly, intraarterially, intraportally or intralesionally; by sustained release system or by implant device. In certain embodiments, the composition can be administered by bolus injection or by continuous infusion or by implant device.

[0655] The composition can also be administered topically via an implant membrane, sponge, or another suitable material onto which the desired molecule is absorbed or encapsulated. In certain embodiments, when an implant device is used, the device can be implanted in any suitable tissue or organ and the desired molecule can be delivered via diffusion, a timed-release bolus, or continuous administration.

[0656] These and other aspects and embodiments of the present invention are described in the accompanying drawings and the following detailed description of the invention and are exemplified in the following examples. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, however, it should be understood that the examples are described in an illustrative and non-limiting manner, and that various modifications may be made by those skilled in the art. Example

[0657] Example 1.1 Production of anti-TROP2 antibodies by hybridoma

[0658] 1.1.1 Immunity

[0659] The human TROP2 extracellular segment protein (Acro, Cat#TR2-H5223) was emulsified with TiterMax (sigma, Cat#T2684) and then immunized Balb / c mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) four times with subcutaneous injection once every two weeks (50 μg protein per mouse).

[0660] 1.1.2 Cell fusion and high-throughput screening

[0661] When the serum titer meets the requirements, the spleen of the mouse is removed to prepare B lymphocyte suspension, which is then electrofused with SP2 / 0 myeloma cells (ATCC). The fused cells are diluted to 1-2×10 cells with selective culture medium (1640 culture medium containing 20% ​​FBS and 1 x HAT). 4 Cells were plated at a concentration of 100 cells / ml in a 96-well plate, and 100 μl of cell suspension was added to each well. On day 7 after fusion, the selection medium (1640 medium containing 10% FBS and 1 x HT) was replaced. After culturing for 10 days (or longer depending on the cell growth status), the supernatant was collected for analysis.

[0662] Hybridoma cells that specifically express anti-TROP2 antibodies were screened by flow cytometry (FACS). The cells to be tested (huTROP2 / GS-CHO & cynoTROP2 / GS-CHO, constructed in-house) were counted and diluted to 1×10 6 For each cell / ml, add 100 μl / well of a U-bottom 96-well plate. Centrifuge at 500 g for 5 minutes and remove the cell culture medium. Add the supernatant from the 96-well hybridoma plate to the U-bottom plate and resuspend the cells. Add 100 μl to each well and incubate on ice for 30 minutes. Centrifuge at 500 g for 5 minutes and remove the supernatant. Wash the cells once with PBS. Centrifuge at 500 g for 5 minutes and remove the PBS. Add 100 μl of FITC-conjugated secondary antibody (1:500 dilution in PBS) against mouse Fab (Jackson Immunoresearch, Cat#115-545-006) to each well. For the positive control antibody, add 100 μl of PE-conjugated secondary antibody against human Fc (Biolegend, Cat#409304). Incubate on ice for 30 minutes in the dark. Remove the supernatant at 500 g for 5 minutes and wash the cells once with PBS. Resuspend the cells in 50 μl of 1× PBS and analyze on a FACS machine. The cell binding positive clones were subjected to affinity test. The final clones were subcloned by limiting dilution method and single clones were picked.

[0663] 1.1.3 Subcloning of positive hybridoma cells

[0664] Limiting dilution subcloning steps: Prepare a 96-well plate and add 200 μl of culture medium to each well. This culture medium is based on the screening medium, but HAT is replaced with HT (Gibco, Cat#11067-030). The rest of the formula is the same. Prepare a cell suspension from the cells in the positive wells screened for fusion. Take 100 μl of each well and add it to the first row. Mix thoroughly. Then take 100 μl of the cell suspension from the first row and add it to the second row. After thorough mixing, take 100 μl and add it to the next row. Repeat the above steps until the last row is diluted. Let the 96-well plate stand for 30 minutes and observe and count under a microscope. Take the volume corresponding to 100 cells and add 20 ml of culture medium. Mix well and plate, 200 μl per well. Observe under a microscope one week later to determine and mark the single clone wells.

[0665] When the confluence of cells in each well reaches more than 50%, the above-mentioned high-throughput screening method is used for detection, the target positive wells are picked out, the cells are frozen after expansion culture, and the clone supernatant is subjected to affinity measurement. The affinity determination method (ForteBio) is carried out according to the existing method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5 (2): p. 270-8). In brief, the AHQ (Pall, 1506091) sensor is offline balanced in the analysis buffer for 30 minutes, and then online detection is performed for 60 seconds to establish a baseline. The purified antibody obtained as described above is loaded online onto the AHQ sensor (ForteBio) for ForteBio affinity measurement. The sensor with the loaded antibody is then exposed to the antigen TROP2, and the sensor is then transferred to the analysis buffer for dissociation rate measurement. K is analyzed using ForteBio analysis software D The affinity test results are shown in Table 1:

[0666] Table 1. Binding kinetic constants of TROP2 hybridoma candidate clones

[0667] Example 1.2 Preparation of chimeric antibodies

[0668] The antibody light and heavy chain gene sequences were retrieved from the hybridoma candidate clone obtained in Example 1.1, and human-mouse chimeric antibodies were constructed. The heavy and light chain sequences of the chimeric antibodies are shown in SEQ ID NOs: 15 and 16, respectively.

[0669] Take about 5×10 cells of each freshly cultured cell line 6 RNA was extracted from each strain (Macherey-Nagel, Cat# 740984.250). cDNA was obtained by reverse transcription using the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara). Upstream primers were designed based on base sequences in the 5' FR1 region, and downstream primers were designed based on bases in the antibody constant region or FR4 region to amplify the antibody light chain and heavy chain variable region gene fragments. These fragments were ligated into a T-vector (Mighty TA-cloning Kit, Takara), and single clones were selected for sequencing. The sequencing results were analyzed and compared using MEGA7 software.

[0670] After comparison, the antibody light and heavy chain variable region sequences were correct and matched clones, and their light and heavy chain variable region gene fragments were respectively passed through the homologous recombinase ( Catalog number: C112-01) was connected to the pcDNA3.1 vector, wherein the constant region selected the IgG1 subtype to obtain the expression plasmids of light chain and heavy chain antibodies.

[0671] The light chain plasmid and heavy chain plasmid of the same antibody were then mixed at a molar ratio of 1:1 and transfected into 293F cells using polyethyleneimine (PEI) (Polysciences, Cat#23966). After 5-7 days of culture, when the cell viability was lower than 60%, the cell culture supernatant was collected and the monoclonal antibody was purified using a Protein A affinity column.

[0672] Example 1.3 In vitro screening of chimeric antibodies

[0673] 1.3.1 Affinity determination

[0674] The equilibrium dissociation constant (K) of the antibody of the present invention binding to human, monkey and mouse TROP2 was determined by thin-layer interferometry (ForteBio). D The ForteBio affinity assay was performed according to the existing method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013. 5(2): p. 270-8).

[0675] Briefly, the AHQ (Pall, 1506091) sensor was equilibrated offline in assay buffer for 30 minutes, followed by online detection for 60 seconds to establish a baseline. The purified antibody obtained as described above was loaded online onto the AHQ sensor (ForteBio) for ForteBio affinity measurement. The sensor with the loaded antibody was then exposed to the antigen TROP2, after which the sensor was transferred to assay buffer for off-rate measurement. K was analyzed using ForteBio analysis software. D Antigens were derived from: human (Acro, Cat#TR2-H5223), monkey (Sino, Cat#50922-M08H), and mouse (Sino, Cat#90893-C08H).

[0676] The test results of antibody affinity are shown in Table 2:

[0677] Table 2. Binding kinetic constants of TROP2 chimeric antibodies

[0678] 1.3.2 Antibody-dependent cellular phagocytosis (ADCP) functional assay

[0679] The present invention uses FcγRIIa-H reporter cells (G9871, Promega) to detect ADCP (antibody-dependent cell-mediated phagocytosis) activity of TROP2 chimeric antibodies. These cells are engineered Jurkat T cells. When FcγRIIa on the cell surface is bound and activated, luciferase is released into the experimental system through the downstream signal NFAT (Nuclear Factor of Activated T), thereby detecting the degree of cell activation.

[0680] The specific method is as follows: a 96-well white flat-bottom cell culture plate was used, and 6×10 target cells FaDu (HTB-43, ATCC) or COLO 205 (TCHu102, Cell Bank of the Chinese Academy of Sciences) or SK-BR-3 (HTB-30, ATCC) were added to each well. 4 and 3×10 effector FcγRIIa-H cells 4 Then, the corresponding concentration of TROP2 monoclonal antibody was added and the cells were cultured in a 37°C incubator for 6 hours. The antibody portion hRS7 of the TROP2 ADC Trodelvy was used as a control antibody (sequence and source see WO2003 / 074566). The culture plate was then removed, Bio-Glo (G7940, Promega) was added, and wavelength detection was performed using a microplate reader (Spectra, Molecular Devices). The results are shown in Figures 1A (FaDu), 1B (COLO 205), and 1C (SK-BR-3). Whether in the TROP2-high-expressing cell line FaDu or the TROP2-moderately-expressing cell lines COLO 205 and SK-BR-3, the ADCP activity of clone ch1D9B10 was superior to that of hRS7, making it suitable for the preparation of ISACs (Immune-Stimulating Antibody Conjugates).

[0681] hRS7 antibody preparation:

[0682] Plasmid DNA for antibody expression was obtained according to WO2003 / 074566.

[0683] Expi293F cells (purchased from Gibco) were cultured with Expi293F medium (Gibco, REF#A14351-01). The cell density was checked one day before transfection (viability should be greater than 95%) and adjusted to 3 × 10 6 The cell density was adjusted to 3 × 10 cells / ml on the day of transfection. 6 cells / ml.

[0684] Take 1 / 10 of the final transfection volume of Opti-MEM medium (Gibco, REF#31985-070) as the transfection buffer, add the DNA to be transfected at a ratio of 1 mg / L, where the light and heavy chain plasmids are in a 1:1 ratio, mix well, add PEIMax (Polysciences Inc. Cat#24765-1) at a DNA:PEI mass ratio of 1:3, mix well, incubate at room temperature for 20 minutes, and then gently pour the mixture into the Expi293F cell suspension while shaking. The cells are cultured in a shaker under the conditions of 8% CO2, 36.5°C, and 120 rpm.

[0685] After 16-18 hours of culture, the cell suspension was supplemented with 2% (v / v) of 200 g / L feed (100 g / L Phytone Peptone + 100 g / L Difco Select Phytone), a glucose solution to a final concentration of 5 g / L, and valproic acid sodium salt (Merk, Cat# P4543-100G) to a final concentration of 2.2 mM. The suspension was gently mixed and cultured for 7 days at 8% CO₂, 36.5°C, and 120 rpm before sampling. The cell suspension was then mixed with diatomaceous earth (Sartorius, Cat 1000037025) (40 g diatomaceous earth per 1 L of cell suspension) and filtered using a 0.22 μm disposable vacuum filter.

[0686] Affinity chromatography to purify the target protein: A HiTrap MabSelect PrismA (GE Healthcare, Cat#17549853) affinity chromatography column was used for affinity capture. Before purification, 10-20 column volumes of 0.1 M NaOH were passed through the tubing and affinity chromatography column, and then the tubing and column were washed with 10-20 column volumes of distilled water. The packed column was equilibrated with 5 column volumes of 1× PBS (Gibco); the filtered cell feed was passed through the column, and the packed column was washed with 10 column volumes of 1× PBS to remove non-specific binding proteins; the packed column was rinsed with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5), the eluate was collected, the pH was adjusted to 6.0 with 2 M Tris, and the column was sterilized by filtration. It was used after passing the purity test.

[0687] Table 3. VH and VL sequences of the final cloned mouse antibodies

[0688] Example 1.4 Humanization of chimeric antibodies

[0689] According to conventional methods, the chimeric antibody obtained by hybridoma is humanized. The specific steps are as follows:

[0690] ① Determine the CDR loop structure;

[0691] ② Find the closest homologous sequence for each V / J region of the heavy and light chains in the human germline sequence database;

[0692] ③ Screening for the human germline that best matches the heavy and light chains and the lowest amount of back mutations;

[0693] ④ Constructing the CDR region of the chimeric antibody onto the human framework region;

[0694] ⑤ Using sequence and structural features, determine the amino acid positions in the framework region that maintain CDR function;

[0695] ⑥ Perform back mutation at the sequence position determined to be important (return to the input amino acid type);

[0696] ⑦Optimize amino acids at risk sites.

[0697] Thus, a humanized antibody was obtained: hz1D9B10, whose CDR sequences, light chain variable regions, heavy chain variable regions, and light chain and heavy chain amino acid sequences are shown in Table 4 below.

[0698] Table 4. Final humanized clone sequences

[0699] Example 1.5 ForteBio determination of affinity between humanized antibodies and antigens

[0700] The equilibrium dissociation constant (KD) of the antibody of the present invention binding to human TROP2 was determined using thin-layer biofilm interferometry (ForteBio). The ForteBio affinity assay follows the existing method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): p. 270-8). In brief, the AHC (Sartorius, 18-5060) sensor was offline balanced in the analysis buffer for 30 minutes, then online for 60 seconds to establish a baseline, and the purified antibody obtained as described above was loaded online onto the AHC sensor (ForteBio) for ForteBio affinity measurement. The sensor with the loaded antibody was then exposed to the antigen human TROP2, after which the sensor was transferred to the analysis buffer for dissociation rate measurement. The KD value was analyzed using ForteBio analysis software.

[0701] The results of antibody affinity test are shown in Table 5. The humanized antibody still has a high affinity for the antigen TROP2, and its equilibrium dissociation constant K is comparable to that of the corresponding chimeric antibody. D .

[0702] Table 5. Affinity constants (M) for antigen-antibody binding assays performed by ForteBio

[0703] Example 1.6 Antibody-dependent cellular phagocytosis (ADCP) functional assay of humanized antibodies

[0704] The ADCP activity of the humanized TROP2 antibody was detected using FcγRIIa-H reporter cells. The target cells were the TROP2-low expressing cell line NUGC-4 (JCRB0834, JCRB CELL BANK). The detection method was the same as in Example 1.3.2. The results are shown in Figure 2. The ADCP activity of hz1D9B10 was comparable to that of the corresponding chimeric antibody and was superior to that of the control antibody hRS7, indicating that the humanization process did not affect the ADCP function of the antibody.

[0705] Example 1.7 Verification of macrophage endocytosis function of humanized antibodies

[0706] The ADCP activity of the humanized clone of the TROP2 antibody was verified using induced differentiated human M1 macrophages. The macrophages and target cells were fluorescently labeled separately. When the two underwent ADCP with the help of the antibody, a double-positive cell population could be observed.

[0707] M1 macrophage differentiation was induced: Monocytes were isolated from fresh human peripheral blood mononuclear cells (PBMCs, TPCS) using a human monocyte enrichment kit (19058, STEMCELL) without CD16 depletion. 6 Resuspend in AIM at a density of 1000 ng / ml. Medium CTS (A3021002, Gibco) was placed in a cell culture flask and cultured at 37°C for 4 hours. Culture medium CTS (A3021002, Gibco) + 10% inactivated fetal bovine serum (FBS, SH30406.05, Hyclone) + 25ng / ml human MCSF1 (216-MC-500, R&D). For the next seven days, culture medium AIM was used every three days. Medium CTS (A3021002, Gibco) + 10% inactivated fetal bovine serum (FBS, SH30406.05, Hyclone) was replaced halfway once. After seven days, medium AIM was used. The medium was half-changed with Medium CTS (A3021002, Gibco) + 10% inactivated fetal bovine serum (FBS, SH30406.05, Hyclone) + 100 ng / ml IFNg (285-IF-100 / CF, R&D), and the macrophage endocytosis experiment was performed the next day.

[0708] Macrophage endocytosis assay: After differentiation, macrophages were digested with Accutase (A6964-500ML, Sigma) and the cell density was adjusted using RPMI-1640 complete medium (22400-071, Gibco) containing 10% fetal bovine serum (FBS, SH30406.05, Hyclone). 1×10 5 The target cells FaDu (HTB-43, ATCC) were labeled with CFSE (C34554, INVITROGEN) at 37°C for 10 min, washed twice with RPMI-1640 complete medium (22400-071, Gibco) containing 10% fetal bovine serum (FBS, SH30406.05, Hyclone), and 2.5×10 4 Target cells and macrophages were mixed with gradiently diluted TROP2 humanized antibodies by pipetting and incubated at 37°C for 4 hours.

[0709] Flow cytometry: Macrophages were labeled with CD14 PE / Cy7 (557742, BD) and then subjected to flow cytometry. Cells in the PE / Cy7 and CFSE double-positive area were identified as the target cell population.

[0710] The results are shown in FIG3 . In the presence of human pharyngeal squamous cell carcinoma cells FaDu, hz1D9B10 antibody can induce ADCP in M1 macrophages, and the activity is comparable to that of the corresponding chimeric antibody.

[0711] Example 2 Synthesis of ISAC molecules

[0712] Example 2.1 Synthesis of Compound 1, Compound 2 and Compound 3

[0713] Synthesis route

[0714] (S)-tert-Butyl (1-((2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl)amino)-1-oxopropan-2-yl)carbamate (1-1)

[0715] To a DMF (12 mL) solution of resiquimod (1.0 g, 3.18 mmol) was added Boc-Ala-OH (0.9 g, 4.77 mmol), DMAP (0.7 g, 5.73 mmol) and EDCI (1.1 g, 5.73 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc (50 mL) and washed successively with a saturated NH4Cl aqueous solution, brine and dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound 1-1 (0.9 g, 58% yield) as a light yellow oil.

[0716] LC-MS (ESI): m / z 486.3 [M+H] +

[0717] (S)-2-amino-N-(2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl)propionamide (1-2)

[0718] To a solution of compound 1-1 (70 mg, 0.144 mmol) in DCM (5 mL) was added TFA (5 mL) at 0° C. The reaction mixture was stirred for 1 hour and then concentrated under reduced pressure to give compound 1-2 (70 mg), which was used in the next step without further purification.

[0719] LC-MS (ESI): m / z 386.2 [M+H] +

[0720] Tert-butyl ((S)-1-(((S)-1-((2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (1-3)

[0721] To a solution of compound 1-2 (70 mg, 0.144 mmol) in DCM (10 mL) was added Boc-Val-OH (32.8 mg, 0.152 mmol), HOBt (22 mg, 0.16 mmol), DIPEA (46.4 mg, 0.36 mmol) and EDCI (30.4 mg, 0.16 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc (30 mL) and washed successively with saturated NH4Cl aqueous solution, brine and dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound 1-3 (50 mg, two-step yield 38%) as a light yellow oil.

[0722] LC-MS (ESI): m / z 585.6 [M+H] + .

[0723] (S)-2-amino-N-((S)-1-((2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4)-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (1-4)

[0724] To a solution of compound 1-3 (200 mg, 0.34 mmol) in DCM (10 mL) was added TFA (10 mL) at 0° C. The reaction mixture was stirred for 1 hour and then concentrated under reduced pressure to give compound 1-4 (200 mg), which was used in the next step without further purification.

[0725] LC-MS (ESI): m / z 485.3 [M+H] + .

[0726] 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (Compound 1)

[0727] To a solution of compound 1-4 (200 mg, 0.34 mmol) in DMF (5 mL) was added N-succinimidyl 6-maleimidocaproate (105 mg, 0.34 mmol) and DIPEA (132 mg, 1.02 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc (20 mL) and washed successively with saturated NH4Cl aqueous solution, brine and dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by preparative HPLC to give compound 1 (65 mg, two-step yield 28%) as a white solid.

[0728] LC-MS (ESI): m / z 678.3 [M+H] + .

[0729] 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12-tetraoxopentadecane-15-carboxamide (Compound 2)

[0730] To a solution of compound 1-4 (82 mg, 0.17 mmol) and Mal-PEG4-acid (59 mg) in DCM (5 mL) was added HATU (65 mg, 0.17 mmol) and DIPEA (55 mg, 0.43 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was washed sequentially with saturated NaHCO3 aqueous solution (5 mL), saturated NH4Cl aqueous solution, and brine and dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by preparative HPLC to give compound 2 (37 mg, 27% yield) as a white solid.

[0731] LC-MS (ESI): m / z 812.2 [M+H] + .

[0732] 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24-octaoxoheptacosa-27-amide (Compound 3)

[0733] To a solution of compound 1-4 (107 mg, 0.22 mmol) and Mal-PEG8-acid (116 mg) in DCM (8 mL) was added HATU (85 mg, 0.22 mmol) and DIPEA (72 mg, 0.56 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was washed sequentially with saturated NaHCO3 aqueous solution (8 mL), saturated NH4Cl aqueous solution, and brine and dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by preparative HPLC to give compound 3 (11 mg, 5% yield) as a white solid.

[0734] LC-MS (ESI): m / z 989.0 [M+H] + .

[0735] Example 2.2 Synthesis of Compound 4

[0736] Synthesis route

[0737] 1-(2-(Ethoxymethyl)-4-(tritylamino)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol (2-1)

[0738] Under N2 atmosphere, Et3N (76 mg, 0.75 mmol) and trityl chloride (100 mg, 0.36 mmol) were added to a suspension of resiquimod (0.1 g, 0.3 mmol) in CH3CN (5 mL) under N2 atmosphere. The reaction mixture was irradiated at 100 ° C for 30 minutes in a microwave reactor. The mixture was cooled to 0 ° C. The precipitate was collected by filtration and washed with cold CH3CN to give compound 2-1 (120 mg, 68% yield) as a white solid.

[0739] LC-MS (ESI): m / z 557.5 [M+H] + .

[0740] N-(tert-Butoxycarbonyl)-(2-((1-(2-(ethoxymethyl)-4-(tritylamino)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)aminosulfonic acid (2-2)

[0741] Under N2 atmosphere, a solution of compound 2-1 (200 mg, 0.36 mmol) in DMF (2 mL) was added dropwise to a suspension of NaH (60% dispersion in oil, 28 mg, 0.72 mmol) in DMF (2 mL). The mixture was stirred at 0 ° C for 1 hour and at room temperature for 30 minutes. 2,2-dioxide-1,2,3-oxathiazolidine-3-carboxylic acid-1,1-dimethylethyl ester (160 mg, 0.72 mmol) was added to the solution at 0 ° C. The mixture was allowed to warm to room temperature and stirred overnight. The mixture was poured into ice water and extracted with EtOAc. The organic layers were combined, washed with water and brine, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH = 150: 1) to obtain compound 2-2 (150 mg, yield 54%) as a white foam.

[0742] LC-MS(ESI):m / z 802.6[M+Na] + .

[0743] 1-(2-(2-aminoethoxy)-2-methylpropyl)-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4-amine (2-3)

[0744] To a solution of compound 2-2 (150 mg, 0.19 mmol) in DCM (8 mL) was added Et3SiH (100 mg) at 0°C, followed by TFA (8 mL). The reaction mixture was stirred at room temperature for 3 hours. The mixture was concentrated under reduced pressure to afford compound 2-3 (67 mg, 97% yield).

[0745] LC-MS (ESI): m / z 358.4 [M+H] + .

[0746] (S)-Benzyl (1-((2-((1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)amino)-4,4-dimethyl-1-oxopentan-2-yl)carbamate (2-4)

[0747] To a solution of compound 2-3 (2.7 g, 7.55 mmol) in DMF (15 mL) at 0°C, DIPEA (3.9 g, 30.2 mmol) and 2,5-dioxopyrrolidin-1-yl (S)-2-(((benzyloxy)carbonyl)amino)-4,4-dimethylpentanoate (3.4 g, 9.06 mmol) were added. The reaction mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc, washed with 0.5N HCl, brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by preparative HPLC to give compound 2-4 (2.5 g, 54% yield) as a white foam.

[0748] LC-MS (ESI): m / z 619.5 [M+H] + .

[0749] (S)-2-amino-N-(2-((1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)-4,4-dimethylpentanamide (2-5)

[0750] Under N2 atmosphere, 10% Pd / C (10 mg) was added to a solution of compound 2-4 (100 mg, 0.16 mmol) in MeOH (5 mL). The reaction mixture was stirred at room temperature under H2 atmosphere for 5 hours. The reaction was filtered and the filtrate was concentrated under reduced pressure to obtain compound 2-5 (NeoR848, 78 mg, quantitative) as a white solid.

[0751] LC-MS (ESI): m / z 485.4 [M+H] + .

[0752] (S)-N-(1-((2-((1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)amino)-4,4-dimethyl-1-oxopentan-2-yl)-1-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxopentadecane-15-carboxamide (Compound 4)

[0753] To a solution of compound 2-5 (78 mg, 0.16 mmol) and Mal-PEG4-acid (56 mg) in DCM (10 mL) was added HATU (61 mg, 0.16 mmol) and DIPEA (41 mg, 0.32 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was washed sequentially with saturated NaHCO3 aqueous solution (5 mL), saturated NH4Cl aqueous solution, and brine and dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by preparative HPLC to give compound 4 (19 mg, 15% yield) in a white foamy form.

[0754] LC-MS (ESI): m / z 812.5 [M+H] + .

[0755] Example 2.3 Synthesis of Compound 5

[0756] Synthesis route

[0757] N-(N-((Benzyloxy)carbonyl)-N-methylglycyl)-N-methylglycine methyl ester (3-1)

[0758] To a solution of Cbz-Sar-OH (10 g, 44.8 mmol) in DCM (100 mL) was added H-Sar-OMe.HCl (6.9 g, 49.3 mmol), DIPEA (14.4 g, 112 mmol), EDCI (9.4 g, 49.3 mmol) and HOBt (6.7 g, 49.3 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was washed sequentially with 1N HCl (3 × 100 mL), water (100 mL), saturated NaHCO3 aqueous solution (3 × 100 mL), brine (100 mL) and dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / EtOAc=100: 1 to 30: 1) to give compound 3-1 (11.2 g, 81% yield) as a light yellow oil.

[0759] LC-MS (ESI): m / z 309.2 [M+H] + .

[0760] N-(N-((Benzyloxy)carbonyl)-N-methylglycyl)-N-methylglycine (3-2)

[0761] To a solution of compound 3-1 (6.0 g, 4.2 mmol) in MeOH (120 mL) was added a solution of LiOH.HO (5.28 g, 126 mmol) in HO (30 mL) at 0 ° C. The reaction mixture was stirred at the same temperature for 2 hours. The mixture was acidified with 3N HCl and extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH=150:1) to give compound 3-2 (5.2 g, 90% yield) as a colorless oil.

[0762] LC-MS (ESI): m / z 317.1 [M+Na] + .

[0763] 4,7,10-trimethyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecane-12-oic acid methyl ester (3-3)

[0764] To a solution of compound 3-2 (5.2 g, 17.67 mmol) in DCM (100 mL) was added H-Sar-OMe.HCl (2.47 g, 17.67 mmol), DIPEA (6.85 g, 53.0 mmol), EDCI (3.74 g, 19.47 mmol) and HOBt (2.63 g, 19.47 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was washed sequentially with 1N HCl (3 × 100 mL), water (100 mL), saturated NaHCO3 aqueous solution (3 × 100 mL), brine (100 mL) and dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / EtOAc=100: 1 to 30: 1) to give compound 3-3 (3.4 g, 51% yield) as a light yellow oil.

[0765] LC-MS (ESI): m / z 402.3 [M+Na] + .

[0766] 4,7,10-Trimethyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecane-12-oic acid (3-4)

[0767] To a solution of compound 3-3 (3.4 g, 8.98 mmol) in MeOH (15 mL) was added a solution of LiOH.HO (1.5 g, 35.9 mmol) in HO (15 mL) at 0 ° C. The reaction mixture was stirred at the same temperature for 2 hours. The mixture was acidified with 3N HCl and extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH=100:1 to 50:1) to give compound 3-4 (2.1 g, yield 64%) as a colorless oil.

[0768] LC-MS (ESI): m / z 388.5 [M+Na] + .

[0769] 4,7,10,13-Tetramethyl-3,6,9,12-tetraoxo-1-phenyl-2-oxa-4,7,10,13-tetraazapentadecan-15-oic acid methyl ester (3-5)

[0770] To a solution of compound 3-4 (3.0 g, 8.2 mmol) in DCM (100 mL) was added H-Sar-OMe.HCl (1.2 g, 8.2 mmol), DIPEA (3.1 g, 24.0 mmol), EDCI (1.7 g, 9.0 mmol) and HOBt (1.2 g, 9.0 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was washed sequentially with 1N HCl (3 × 100 mL), water (100 mL), saturated NaHCO 3 (3 × 100 mL), brine (100 mL) and dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / EtOAc = 100: 1 to 30: 1) to give compound 3-5 (1.2 g, yield 32%) as a light yellow oil. LC-MS (ESI): m / z 451.3 [M + H] + .

[0771] 4,7,10,13-Tetramethyl-3,6,9,12-tetraoxo-1-phenyl-2-oxa-4,7,10,13-tetraazapentadecan-15-oic acid (3-6)

[0772] To a solution of compound 3-5 (12.3 g, 27.3 mmol) in MeOH (120 mL) was added a solution of LiOH.HO (4.6 g, 109.2 mmol) in HO (30 mL) at 0 ° C. The reaction mixture was stirred at the same temperature for 2 hours. The mixture was acidified with 3N HCl and extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH=100:1 to 50:1) to give compound 3-6 (5.3 g, 45% yield) as a colorless oil.

[0773] LC-MS (ESI): m / z 459.2 [M+Na] + .

[0774] (S)-(20-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-3,6,9,12,15-pentamethyl-13-neopentyl-2,5,8,11,14-pentaoxo-18-oxa-3,6,9,12,15-pentaazaeicosyl)(methyl)carbamic acid benzyl ester (3-7)

[0775] At 0 ° C, EDCI (114 mg, 0.60 mmol) and HOBt (80 mg, 0.60 mmol) were added to a DCM (10 mL) solution of compound 3-6 (200 mg, 0.46 mmol). After stirring for 5 minutes, compound 2-5 (Example 2.2, 266 mg, 0.56 mmol) and DIPEA (178 mg, 1.38 mmol) were added. The reaction mixture was stirred at room temperature overnight. The mixture was washed with 1N HCl (2 × 10 mL), water (10 mL), NaHCO (2 × 10 mL), brine (10 mL) and dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by preparative HPLC to obtain compound 3-7 (148 mg, 36% yield) as a light yellow solid.

[0776] LC-MS (ESI): m / z 903.7 [M+H] + .

[0777] (S)-(20-(2-(Ethoxymethyl)-4-(tritylamino)-1H-imidazo[4,5-c]quinolin-1-yl)-3,6,9,12,15-pentamethyl-13-neopentyl-2,5,8,11,14-pentaoxo-18-oxa-3,6,9,12,15-pentaazaeicosyl)(methyl)carbamic acid benzyl ester (3-8)

[0778] Under N2 atmosphere, Et3N (41 mg, 40.5 mmol) and trityl chloride (68 mg, 0.23 mmol) were added to a suspension of compound 3-7 (180 mg, 0.18 mmol) in CH3CN (5 mL) under N2 atmosphere. The reaction mixture was irradiated at 80 ° C for 45 minutes in a microwave reactor. The mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (DCM / MeOH=20:1) to give compound 3-8 (100 mg, 44%) as a yellow solid.

[0779] LC-MS (ESI): m / z 1145.9 [M+H] + .

[0780] (S)-N-(2-((1-(2-(ethoxymethyl)-4-(tritylamino)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)-4,4-dimethyl-2-(5,8,11-trimethyl-4,7,10-trioxo-2,5,8,11-tetraazatridecane-13-amido)pentanamide (3-9)

[0781] Under N2 atmosphere, 10% Pd / C (40 mg) was added to a solution of compound 3-8 (80 mg) in MeOH (5 mL). The reaction mixture was stirred at room temperature under H2 atmosphere for 4 hours. The reaction was filtered and the filtrate was concentrated under reduced pressure to obtain compound 3-9 (69 mg, 98% yield) as a yellow solid.

[0782] LC-MS (ESI): m / z 1011.6 [M+H] + .

[0783] (S)-2-(14-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetramethyl-4,7,10,13-tetraoxo-3,6,9,12-tetraazatetradecanoylamino)-N-(2-((1-(2-(ethoxymethyl)-4-(tritylamino)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylprop-2-yl)oxy)ethyl)-4,4-dimethylpentanamide (3-10)

[0784] To a solution of compound 3-9 (69 mg, 0.069 mmol) and maleimidoacetic acid (13.8 mg, 0.069 mmol) in DCM (25 mL) was added HATU (33 mg, 0.083 mmol) and DIPEA (19 mg, 0.14 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was washed with saturated NaHCO aqueous solution (20 mL). Saturated NH4Cl aqueous solution (20 mL), brine (20 mL) were washed successively and dried with anhydrous Na2SO4, and concentrated under reduced pressure to give compound 3-10 (85 mg) as a yellow solid.

[0785] LC-MS (ESI): m / z 1149.0 [M+H] + .

[0786] (S)-N-(2-((1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)-2-(14-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetramethyl-4,7,10,13-tetraoxo-3,6,9,12-tetraazatetradecanoylamino)-4,4-dimethylpentanamide (Compound 5)

[0787] To a solution of compound 3-10 (85 mg, 0.074 mmol) in DCM (5 mL) was added Et3SiH (18 mg, 0.148 mmol) and TFA (0.5 mL) at 0°C. The reaction mixture was stirred at room temperature for 4 hours. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC to give compound 5 (11 mg, 18% yield over two steps) in the form of a white foam.

[0788] LC-MS (ESI): m / z 906.8 [M+H] + .

[0789] Example 2.4 Synthesis of Compound 6

[0790] Synthesis route

[0791] N-Methyl-N-(N-methyl-N-(N-methyl-N-(methylglycyl)glycyl)glycyl)glycine methyl ester (4-1)

[0792] To a solution of methyl 4,7,10,13-tetramethyl-3,6,9,12-tetraoxo-1-phenyl-2-oxa-4,7,10,13-tetraazapentadecan-15-oate (5 g, 11.1 mmol) in MeOH (50 mL) was added 10% Pd / C (0.5 mg) under N2 atmosphere. The reaction mixture was stirred at room temperature under H2 atmosphere for 6 hours. The reactant was filtered and the filtrate was concentrated under reduced pressure to obtain compound 4-1 (3.5 g, quantitative) as a light yellow oil.

[0793] LC-MS (ESI): m / z 339.1 [M+Na] + .

[0794] 4,7,10,13,16,19,22,25-octamethyl-3,6,9,12,15,18,21,24-octaoxo-1-phenyl-2-oxa-4,7,10,13,16,19,22,25-octaazaheptacosane-27-oic acid methyl ester (4-2)

[0795] To a solution of Cbz-Sar-Sar-Sar-Sar-OH (4.8 g, 11.1 mmol) in DCM (80 mL) was added HATU (5.06 g, 13.3 mmol) at 10 ° C. After stirring for 5 minutes, a solution of H-Sar-Sar-Sar-Sar-OMe (3.5 g, 11.1 mmol) and DIPEA (2.86 g, 22.2 mmol) in DCM (25 mL) was added. The reaction mixture was stirred at room temperature overnight. The mixture was washed sequentially with 1N HCl (100 mL), water (100 mL), saturated NaHCO (100 mL), brine (100 mL) and dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH=100: 1 to 50: 1) to give compound 4-2 (2.2 g, 27% yield) as a light yellow oil.

[0796] LC-MS (ESI): m / z 757.7 [M+Na] + .

[0797] 4,7,10,13,16,19,22,25-Octamethyl-3,6,9,12,15,18,24-octaoxo-1-phenyl-2-oxa-4,7,10,13,16,19,22,25-octaazaheptacosanoic acid (4-3)

[0798] To a solution of compound 4-2 (2.2 g, 2.99 mmol) in MeOH (15 mL) was added a solution of LiOH.H2O (0.5 g, 11.96 mmol) in H2O (5 mL) at 0°C. The reaction mixture was stirred at the same temperature for 3 hours. The solvent was removed by concentration. The resulting aqueous solution was acidified with 3N HCl and washed with EtOAc. Water was removed by freeze-drying. The residue was purified by preparative HPLC to obtain compound 4-3 (601 mg, 28% yield) as a white solid.

[0799] LC-MS (ESI): m / z 721.6 [M+H] + .

[0800] 4,7,10,13,16,19,22,25-Octamethyl-3,6,9,12,15,18,24-octaoxo-1-phenyl-2-oxa-4,7,10,13,16,19,22,25-octaazaheptacosane-27-oic acid tert-butyl ester (4-4)

[0801] To a solution of compound 4-3 (600 mg, 0.83 mmol) in DCM (10 mL) was added tert-butyl N,N'-diisopropylcarbamate (928 mg, 4.63 mmol) at 0°C. The reaction mixture was stirred at room temperature overnight. The mixture was washed sequentially with 1N HCl (2 × 5 mL), water (5 mL), saturated NaHCO3 (2 × 5 mL), and brine (5 mL), and dried over anhydrous Na2SO4. The mixture was concentrated under reduced pressure to give compound 4-4 (702 mg, quantitative) as a light yellow oil.

[0802] LC-MS (ESI): m / z 799.8 [M+H] + .

[0803] 5,8,11,14,17,20,23-Heptamethyl-4,7,10,13,16,19,22-heptaoxo-2,5,8,11,14,17,20,23-octaazapentacosane-25-oic acid tert-butyl ester (4-5)

[0804] Under N2 atmosphere, 10% Pd / C (100 mg) was added to a solution of compound 4-4 (700 mg) in MeOH (10 mL). The reaction mixture was stirred at room temperature under H2 atmosphere for 4 hours. The reaction was filtered and the filtrate was concentrated under reduced pressure to obtain compound 4-5 (360 mg, 62% yield) as a light yellow oil.

[0805] LC-MS (ESI): m / z 643.2 [M+H] + .

[0806] tert-Butyl 26-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15,18,21,24-octamethyl-4,7,10,13,16,19,22,25-octaoxo-3,6,9,12,15,18,21,24-octaazahexacosanoate (4-6)

[0807] To a solution of compound 4-5 (360 mg, 0.56 mmol) and maleimidoacetic acid (104 mg, 0.672 mmol) in DCM (10 mL) was added HATU (255 mg, 0.672 mmol) and DIPEA (108 mg, 0.84 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was washed successively with saturated NaHCO aqueous solution (5 mL), saturated NH4Cl aqueous solution (5 mL), brine (5 mL) and dried over anhydrous Na2SO4, and concentrated under reduced pressure to give compound 4-6 (436 mg, quantitative) in a light yellow oily form.

[0808] LC-MS (ESI): m / z 802.3 [M+H] + .

[0809] 26-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15,18,21,24-octamethyl-4,7,10,13,16,19,22,25-octaoxo-3,6,9,12,15,18,21,24-octaazahexacosanoic acid (4-7)

[0810] To a solution of compound 4-6 (435 mg, 0.56 mmol) in DCM (8 mL) was added TFA (4 mL) at 0 ° C. The reaction mixture was stirred at room temperature for 4 hours. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC to give compound 4-7 (295 mg, 74% yield) as a white solid.

[0811] LC-MS (ESI): m / z 746.4 [M+Na] + .

[0812] (S)-N-(2-((1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)-2-(2,6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15,18,21,24-octamethyl-4,7,10,13,16,19,22,25-octaoxo-3,6,9,12,15,18,21,24-octaazahexacosanoylamino)-4,4-dimethylpentanamide (Compound 6)

[0813] At 0 ° C, EDCI (30 mg, 0.16 mmol) and HOBt (21 mg, 0.16 mmol) were added to a DCM (2 mL) solution of compound 4-7 (90 mg, 0.12 mmol). After stirring for 5 minutes, compound 2-5 (Example 2.2, 59 mg, 0.13 mmol) was added. The reaction mixture was stirred at room temperature for 4 hours. The residue was purified by preparative HPLC to obtain compound 6 (36 mg, 24% yield) as a white solid.

[0814] LC-MS (ESI): m / z 1212.8 [M+Na] + .

[0815] Example 2.5 Synthesis of Compound 7 and Compound 8

[0816] Synthesis route

[0817] (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((S)-12-(4-amino-2-(ethoxymethyl))-1H-imidazo[4,5-c]quinolin-1-yl)-11,11-dimethyl-5-neopentyl-3,6-dioxo-2,10-dioxa-4,7-diazadodecyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (5-2)

[0818] To a solution of compound 2-5 (Example 2.2, 250 mg, 0.516 mmol) and DIPEA (272 mg, 2.1 mmol) in DMF (8 mL) was added Fmoc-Val-Cit-PAB-PNP (396 mg, 0.516 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc (20 mL), washed sequentially with saturated NaHCO3 aqueous solution (5 mL), NH4Cl aqueous solution (5 mL), and brine (5 mL), and dried over anhydrous Na2SO4, and concentrated under reduced pressure to give compound 5-2 (crude, 550 mg). LC-MS (ESI): m / z 1112.1 [M+H] + .

[0819] 4-((S)-2-((S)-2-amino-3-methylbutyrylamino)-5-ureidopentanamido)benzyl((S)-1-((2-((1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)amino)-4,4-dimethyl-1-oxopentan-2-yl)carbamate (5-3)

[0820] To a solution of compound 5-2 (550 mg, 0.19 mmol) in DMF (5 mL) was added piperidine (420 mg) at 0°C. The reaction mixture was stirred at room temperature for 3 hours. The mixture was purified by preparative HPLC to give compound 5-3 (238 mg, 52% yield).

[0821] LC-MS (ESI): m / z 890.3 [M+H] + .

[0822] 4-((2S,5S)-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-9,12,15,18-tetramethyl-4,7,10,13,16,19-hexaoxo-2-(3-ureidopropyl)-3,6,9,12,15,18-hexaazaeicosanoylamino)benzyl((S)-1-((2-((1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)amino)-4,4-dimethyl-1-oxopentan-2-yl)carbamate (Compound 7)

[0823] To a DMF (0.5 mL) solution of Mal-Sar4-OH (25 mg, 0.056 mmol) and HOBt (10 mg, 0.07 mmol) was added EDCI (14 mg, 0.07 mmol). The reaction mixture was stirred at room temperature for 2 hours under N2. The reaction mixture was stirred at room temperature for 10 minutes under N2, and then compound 5-3 (50 mg 0.056 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours under N2. The mixture was filtered and the filtrate was purified by preparative HPLC to obtain compound 7 (20 mg, 27% yield) as a white solid.

[0824] LC-MS (ESI): m / z 1311.3 [M+H] + .

[0825] 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoylamino)-3-methylbutyrylamino)-5-ureidopentanamido)benzyl((S)-1-((2-((1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)amino)-4,4-dimethyl-1-oxopentan-2-yl)carbamate (Compound 8)

[0826] To a DMF (2 mL) solution of compound 5-3 (60 mg, 0.067 mmol) was added DIPEA (8.8 mg, 0.067 mmol) and N-succinimidyl 6-maleimidohexanoate (21 mg, 0.067 mmol). The reaction mixture was stirred at room temperature for 4 hours. The mixture was purified by preparative HPLC to give compound 8 (20 mg, 27% yield) as a white solid. LC-MS (ESI): m / z 1083.2 [M+H] + .

[0827] Example 2.6 Synthesis of Compound 9

[0828] Synthesis route

[0829] Benzyl (1-((2-((1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)amino)-2-methyl-1-oxopropan-2-yl)carbamate (6-4)

[0830] To a solution of compound 2-3 (see Synthesis of Compound 4) (600 mg, 1.68 mmol) in DMF (10 mL) was added DIPEA (759 mg, 5.88 mmol) and 2,5-dioxopyrrolidin-1-yl (S)-2-(((benzyloxy)-carbonyl)amino)-4,4-dimethylpentanoate (573 mg, 1.7 mmol) at 0°C. The reaction mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc (50 mL), washed with 0.5N HCl and brine, dried over anhydrous NaSO, and concentrated under reduced pressure. The residue was purified by preparative HPLC to give compound 6-4 (560 mg, 58% yield) as a white foam.

[0831] LC-MS (ESI): m / z 577.1 [M+H] + .

[0832] 2-amino-N-(2-((1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)-2-methylpropanamide (6-5)

[0833] To a solution of compound 6-4 (560 mg, 0.97 mmol) in MeOH (10 mL) was added 10% Pd / C (500 mg) under N2 atmosphere. The reaction mixture was stirred at room temperature under H2 atmosphere for 4 hours. The reactants were filtered and the filtrate was concentrated under reduced pressure to give compound 6-5 (390 mg, 91% yield) as a white solid.

[0834] LC-MS (ESI): m / z 443.2 [M+H] + .

[0835] (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(12-(4-amino-2-(ethoxymethyl))-1H-imidazo[4,5-c]quinolin-1-yl)-5,5,11,11-tetramethyl-3,6-dioxo-2,10-dioxa-4,7-diazadodecyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (6-6)

[0836] To a solution of compound 6-5 (390 mg, 0.88 mmol) and DIPEA (569 mg, 4.4 mmol) in DMF (10 mL) was added Fmoc-Val-Cit-PAB-PNP (675 mg, 0.88 mmol), and the reaction mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc (50 mL), washed with saturated NaHCO3 aqueous solution (5 mL), NH4Cl aqueous solution (5 mL), and brine (5 mL) in that order, and dried over anhydrous Na2SO4, and concentrated under reduced pressure to give compound 6-6 (crude product, 958 mg).

[0837] LC-MS(ESI):m / z 1070.3[M+H] + .

[0838] 4-((S)-2-((S)-2-amino-3-methylbutyrylamino)-5-ureidopentanamido)benzyl (1-((2-((1-(4-amino-2-(ethoxymethyl))-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)amino)-2-methyl-1-oxopropan-2-yl)carbamate (6-7)

[0839] To a solution of compound 6-6 (958 mg, 0.88 mmol) in DMF (5 mL) was added piperidine (750 mg) at 0°C. The reaction mixture was stirred at room temperature for 3 hours. The mixture was purified by preparative HPLC to give compound 6-7 (210 mg, 28% yield).

[0840] LC-MS (ESI): m / z 848.1 [M+H] + .

[0841] 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoylamino)-3-methylbutyrylamino)-5-ureidopentanamido)benzyl (1-((2-((1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)oxy)ethyl)amino)-2-methyl-1-oxopropan-2-yl)carbamate (Compound 9)

[0842] To a solution of compound 6-7 (50 mg, 0.056 mmol) in DMF (1 mL) was added DIPEA (7 mg, 0.056 mmol) and N-succinimidyl 6-maleimidohexanoate (18 mg, 0.056 mmol), and the reaction mixture was stirred at room temperature for 4 hours. The mixture was purified by preparative HPLC to give compound 9 (15 mg, 25% yield) as a white solid.

[0843] LC-MS(ESI):m / z 1041.1[M+H] + .

[0844] Example 3 Activity Detection of TLR7 / 8 Immune Agonists in H-TLR7 NFKB Reporter 293 and H-TLR8 NFKB Reporter 293 Cell Lines

[0845] The present invention uses H-TLR7 NFKB Reporter 293 (GM-C15426, Yoshiman Bio) and H-TLR8 NFKB Reporter 293 (GM-C15427, Yoshiman Bio) reporter gene cells to detect TLR7 and TLR8 activation activity of TLR7 / 8 immune agonists. When the TLR7 and TLR8 receptors in the cells are bound and activated, luciferase is released into the experimental system through the downstream signal NF-κB, thereby detecting the degree of cell activation.

[0846] The specific method is as follows: a 96-well white flat-bottom cell culture plate was used, and 3×10 H-TLR7 NFKB Reporter 293 (GM-C15426, Jiman Bio) and H-TLR8 NFKB Reporter 293 (GM-C15427, Jiman Bio) reporter gene cells were added to each well. 4 The cells were grown overnight at 37°C. The next day, the supernatant was discarded, and the corresponding concentration of TLR7 / 8 immune agonist was added. The cells were incubated at 37°C for another 6 hours. The culture plates were then removed and Bio-Glo (G7940, Promega) was added. The wavelength was measured using a microplate reader (Spectra, Molecular Devices).

[0847] The TLR7 activity of each compound is shown in Figures 4A and 4B, and the TLR8 activity is shown in Figures 4C and 4D. As can be seen from Figure 4A, all compounds except Compound A1, Compound A3, Compound A6, Compound A11, Compound A8, and Compound A4 can be detected as active on the H-TLR7 NFKB Reporter 293, indicating that only Compound A1, Compound A3, Compound A6, Compound A11, Compound A8, and Compound A4 are not TLR7 agonists, while the other compounds are TLR7 agonists. In addition, different compounds have different activation potencies. For example, Compound A20, Compound A21, Compound A9, Compound A10, Compound A5, Compound A16, and Compound A22 have higher TLR7 activity than Resiquimod (R848), while the TLR7 activity of the remaining compounds is lower than Resiquimod (R848). As can be seen from Figure 4B, the TLR7 activity of compounds A13, A18, and A19 is weak, and compound A17 has no TLR7 activity. As can be seen from Figure 4C, compounds A11, A8, A6, A5, A3, A1, and A20 showed high activity on H-TLR8 NFKB Reporter 293, which was higher than that of Resiquimod (R848). The TLR8 activity of compounds A21 and A10 was lower than that of Resiquimod (R848), and the remaining compounds had no TLR8 activity. As can be seen from Figure 4D, compounds A13, A17, A18, and A19 all had no TLR8 activity.

[0848] Therefore, Compound A2, Compound A12, Compound A14, Compound A15, Compound A16, Compound A9, Compound A22, Compound A23, Compound A24, Compound A25, Compound A13, Compound A18, and Compound A19 are TLR7 agonists, Compound A1, Compound A3, Compound A6, Compound A11, and Compound A8 are TLR8 agonists, and Compound A5, Compound A20, Compound A21, and Compound A10 are TLR7 / 8 dual agonists, among which Compound A5 is a TLR7 / 8 dual agonist that is more active than Resiquimod (R848).

[0849] Example 4 Preparation of Conjugate

[0850] 4.1 Preparation of hz1D9B10-compound 5

[0851] The specific method is as follows:

[0852] (a) Antibody hz1D9B10 was dissolved in PBS buffer.

[0853] (b) Add a reducing agent solution (TCEP, Aldrich, Catalog Number 646547, dissolved in water) and allow the reaction mixture to react at room temperature for 2 hours.

[0854] (i) The optimal concentration of hz1D9B10 is 5-10 mg / mL,

[0855] (ii) The optimal molar ratio of TCEP / mAb is 2.0,

[0856] (iii) The optimal reaction temperature is 25°C.

[0857] (iv) The optimal pH value of the reaction is between 6.0 and 8.0.

[0858] (c) adding an excess of linker-toxin (compound 5, dissolved in DMSO) to react with the antibody reduced in step (b), and the reaction mixture was placed at room temperature for 2 hours, wherein

[0859] (i) The optimal molar ratio of compound 5 / mAb is 6.0,

[0860] (ii) The optimal reaction temperature is 25°C,

[0861] The crude ADC product was obtained.

[0862] (d) The obtained crude ADC product is purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product.

[0863] (e) The ADC product was tested using RP-HPLC, LC-MS, and SEC HPLC to determine the average DAR of 4 and SEC purity.

[0864] 4.2 Preparation of hz1D9B10-compound 6

[0865] The specific method is as follows:

[0866] (a) Antibody hz1D9B10 was dissolved in PBS buffer.

[0867] (b) Add a reducing agent solution (TCEP, Aldrich, Catalog Number 646547, dissolved in water) and allow the reaction mixture to react at room temperature for 2 hours.

[0868] (i) The optimal concentration of hz1D9B10 is 5-10 mg / mL,

[0869] (ii) The optimal molar ratio of TCEP / mAb is 2.0,

[0870] (iii) The optimal reaction temperature is 25°C.

[0871] (iv) The optimal pH value of the reaction is between 6.0 and 8.0.

[0872] (c) adding an excess of linker-toxin (compound 6, dissolved in DMSO) to react with the antibody reduced in step (b), and the reaction mixture was placed at room temperature for 2 hours, wherein

[0873] (i) The optimal molar ratio of compound 6 / mAb is 6.0,

[0874] (ii) The optimal reaction temperature is 25°C,

[0875] The crude ADC product was obtained.

[0876] (d) The obtained crude ADC product is purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product.

[0877] (e) The ADC product was tested using RP-HPLC, LC-MS, and SEC HPLC to determine the average DAR of 4 and SEC purity.

[0878] 4.3 Preparation of hz1D9B10-compound 7

[0879] The specific method is as follows:

[0880] (a) Antibody hz1D9B10 was dissolved in PBS buffer.

[0881] (b) Add a reducing agent solution (TCEP, Aldrich, Catalog Number 646547, dissolved in water) and allow the reaction mixture to react at room temperature for 2 hours.

[0882] (i) The optimal concentration of hz1D9B10 is 5-10 mg / mL,

[0883] (ii) The optimal molar ratio of TCEP / mAb is 2.0,

[0884] (iii) The optimal reaction temperature is 25°C.

[0885] (iv) The optimal pH value of the reaction is between 6.0 and 8.0.

[0886] (c) adding an excess of linker-toxin (compound 7, dissolved in DMSO) to react with the antibody reduced in step (b), and the reaction mixture was placed at room temperature for 2 hours, wherein

[0887] (i) The optimal molar ratio of compound 7 / mAb is 6.0,

[0888] (ii) The optimal reaction temperature is 25°C,

[0889] The crude ADC product was obtained.

[0890] (d) The obtained crude ADC product is purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product.

[0891] (e) The ADC product was tested using RP-HPLC, LC-MS, and SEC HPLC to determine the average DAR of 4 and SEC purity.

[0892] 4.4 Preparation of hz1D9B10-Compound 8

[0893] The specific method is as follows:

[0894] (a) Antibody hz1D9B10 was dissolved in PBS buffer.

[0895] (b) Add a reducing agent solution (TCEP, Aldrich, Catalog Number 646547, dissolved in water) and allow the reaction mixture to react at room temperature for 2 hours. (i) The optimal concentration of hz1D9B10 is 5-10 mg / mL.

[0896] (ii) The optimal molar ratio of TCEP / mAb is 2.0,

[0897] (iii) The optimal reaction temperature is 25°C.

[0898] (iv) The optimal pH value of the reaction is between 6.0 and 8.0.

[0899] (c) adding an excess of linker-toxin (compound 8, dissolved in DMSO) to react with the antibody reduced in step (b), and the reaction mixture was placed at room temperature for 2 hours, wherein

[0900] (i) The optimal molar ratio of compound 8 / mAb is 6.0,

[0901] (ii) The optimal reaction temperature is 25°C,

[0902] The crude ADC product was obtained.

[0903] (d) The obtained crude ADC product is purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product.

[0904] (e) The ADC product was tested using RP-HPLC, LC-MS, and SEC HPLC to determine the average DAR of 4 and SEC purity.

[0905] 4.5 Preparation of hz1D9B10-Compound 9

[0906] The specific method is as follows:

[0907] (a) Antibody hz1D9B10 was dissolved in PBS buffer.

[0908] (b) Add a reducing agent solution (TCEP, Aldrich, Catalog Number 646547, dissolved in water) and allow the reaction mixture to react at room temperature for 2 hours.

[0909] (i) The optimal concentration of hz1D9B10 is 5-10 mg / mL,

[0910] (ii) The optimal molar ratio of TCEP / mAb is 2.0,

[0911] (iii) The optimal reaction temperature is 25°C.

[0912] (iv) The optimal pH value of the reaction is between 6.0 and 8.0.

[0913] (c) adding an excess of linker-toxin (compound 9, dissolved in DMSO) to react with the antibody reduced in step (b), and the reaction mixture was placed at room temperature for 2 hours, wherein

[0914] (i) The optimal molar ratio of compound 9 / mAb is 6.0,

[0915] (ii) The optimal reaction temperature is 25°C,

[0916] The crude ADC product was obtained.

[0917] (d) The obtained crude ADC product is purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product.

[0918] (e) The ADC product was tested using RP-HPLC, LC-MS, and SEC HPLC to determine the average DAR of 4 and SEC purity.

[0919] 4.6 Preparation of IgG-Compound 6

[0920] The specific method is as follows:

[0921] (a) Antibody isotype control IgG1 was dissolved in PBS buffer.

[0922] (b) Add a reducing agent solution (TCEP, Aldrich, Catalog Number 646547, dissolved in water) and allow the reaction mixture to react at room temperature for 2 hours.

[0923] (i) The optimal concentration of isotype control IgG1 is 5-10 mg / mL,

[0924] (ii) The optimal molar ratio of TCEP / mAb is 2.0,

[0925] (iii) The optimal reaction temperature is 25°C.

[0926] (iv) The optimal pH value of the reaction is between 6.0 and 8.0.

[0927] (c) adding an excess of linker-toxin (compound 6, dissolved in DMSO) to react with the antibody reduced in step (b), and the reaction mixture was placed at room temperature for 2 hours, wherein

[0928] (i) The optimal molar ratio of compound 6 / mAb is 6.0,

[0929] (ii) The optimal reaction temperature is 25°C,

[0930] The crude ADC product was obtained.

[0931] (d) The obtained crude ADC product is purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product.

[0932] (e) The ADC product was tested using RP-HPLC, LC-MS, and SEC HPLC to determine the average DAR of 4 and SEC purity.

[0933] 4.7 Preparation of IgG-NT3

[0934] The specific method is as follows:

[0935] (a) Antibody isotype control IgG1 was dissolved in PBS buffer.

[0936] (b) Add a reducing agent solution (TCEP, Aldrich, Catalog Number 646547, dissolved in water) and allow the reaction mixture to react at room temperature for 2 hours.

[0937] (i) The optimal concentration of isotype control IgG1 is 5-10 mg / mL,

[0938] (ii) The optimal molar ratio of TCEP / mAb is 2.0,

[0939] (iii) The optimal reaction temperature is 25°C.

[0940] (iv) The optimal pH value of the reaction is between 6.0 and 8.0.

[0941] (c) adding an excess of linker-toxin (NT3, from WO2021173773A1, dissolved in DMSO) to react with the antibody reduced in step (b), and the reaction mixture was placed at room temperature for 2 hours, wherein

[0942] (i) The optimal molar ratio of NT3 / mAb is 6.0,

[0943] (ii) The optimal reaction temperature is 25°C,

[0944] The crude ADC product was obtained.

[0945] (d) The obtained crude ADC product is purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product.

[0946] (e) The ADC product was tested using RP-HPLC, LC-MS, and SEC HPLC to determine the average DAR of 4 and SEC purity.

[0947] 4.8 Preparation of hRS7-NT3

[0948] hRS7-NT3 of the following formula was prepared by the following method

[0949] wherein A is hRS7 and p' is about 4.3.

[0950] (a) Dissolve the hRS7 antibody in PBS buffer (pH 7.4);

[0951] (b) adding a reducing agent solution (TCEP, Aldrich, Catalog Number 646547, dissolved in water), and incubating the reaction mixture at room temperature for 2 hours, wherein: (i) the optimal concentration of hRS7 is 5 mg / mL, (ii) the optimal molar ratio of TCEP / mAb is 2.0, (iii) the optimal reaction temperature is 25°C, and (iv) the optimal reaction pH is between 6.0 and 8.0;

[0952] (c) adding an excess of NT3 (from WO2021173773A1, dissolved in DMSO) to react with the antibody reduced in step (a), and the reaction mixture was placed at room temperature for 2 hours, wherein: (i) the optimal molar ratio of NT3 / mAb was 6.0,

[0953] (ii) The optimal reaction temperature is 25° C., thereby obtaining a crude ADC product;

[0954] (d) the obtained crude ADC product is purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product;

[0955] (e) ADC products were tested using RP-HPLC, LC-MS, and SEC HPLC to determine the average DAR and SEC purity.

[0956] Among them, the DAR average value was 4.3 and the SEC purity was 98.42.

[0957] Example 5 Activity of Different Antibody-Stimulating Antibody Conjugates (ISACs) in PBMC and Tumor Cell Co-culture System

[0958] Monocytes were isolated from human peripheral blood mononuclear cells (PBMC, TPCS) using a human monocyte enrichment kit without CD16 depletion (19058, STEMCELL) and the number of cells was 1 × 10 7 The cells were resuspended at a density of 100 cells / ml in RPMI-1640 complete medium (22400-071, Gibco) containing 10% fetal bovine serum (FBS, SH30406.05, Hyclone) and 0.1% β-mercaptoethanol (21985023, Gibco), and 5×10 cells / ml were added to each well of a 96-well flat-bottom cell culture plate. 5 Digest target FaDu cells (HTB-43, ATCC) and adjust the cell density to 2×10 6 1×10 / ml was added to each well. 5The cells were then treated with the corresponding concentrations of TROP2 ISAC or TROP2 monoclonal antibody, mixed evenly, and incubated at 37°C for 18 hours. The cell culture plate was removed, and IFNα secretion in the cell-free supernatant was assayed using ELISA. As shown in Figure 5, the IFNα secretion levels under the action of hz1D9B10-compound 6, hz1D9B10-compound 7, and hz1D9B10-compound 8 were all lower than those under the action of hz1D9B10-compound 9, indicating that the in vitro activity of hz1D9B10-compound 6, hz1D9B10-compound 7, and hz1D9B10-compound 8 was weaker than that of hz1D9B10-compound 9.

[0959] Example 6 Therapeutic Effects of TROP2-ISAC in the NCI-N87 Model

[0960] 6.1 Therapeutic Effects of hz1D9B10-Compound 7 and hz1D9B10-Compound 9 in the NCI-N87 Model

[0961] In this experiment, NCI-N87 cells were inoculated into CB17-SCID mice to determine the anti-tumor effect of the TROP2-ISAC antibody of the present invention.

[0962] CB17-SCID mice:

[0963] Female CB17-SCID mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. They were SPF grade and quality-inspected by Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., with a certificate number of 110011221100703563. Upon arrival, the mice were quarantined for 3 days before the study began.

[0964] cell:

[0965] NCI-N87 cells were purchased from ATCC (CAT#:CRL-5822) and routine subculture was performed in strict accordance with the instructions for subsequent in vivo experiments. Cells were collected by centrifugation, resuspended in sterile PBS, and the cell density was adjusted to 10×10 6 On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal area of ​​CB17-SCID mice to establish the NCI-N87 tumor-bearing mouse model.

[0966] Dosage:

[0967] Eight days after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with tumor volumes of approximately 78-138 mm were selected. 3The mice were divided into groups according to the tumor volume (6 mice per group). The dosage and method of administration are shown in Table 8. h-IgG (purchased from EQUITECH-BIO) was used as a negative control and administered on the 8th day after inoculation. The tumor volume and body weight of the mice were monitored twice a week. The body weight and tumor volume were measured before each administration. The relative tumor inhibition rate (TGI%) was calculated on the 26th day after inoculation. The calculation formula is as follows: TGI% = 100% * (tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume of the control group before administration). Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper. The tumor volume was calculated according to the following formula: V = L × W 2 / 2. Body weight was measured using an electronic balance.

[0968] Table 8. Experimental design

[0969] The tumor inhibition rates for the hz1D9B10, hz1D9B10-Compound 7, and hz1D9B10-Compound 9 groups were 8.44%, 104%, and 113%, respectively. hz1D9B10-Compound 7 and hz1D9B10-Compound 9 demonstrated comparable tumor inhibition, significantly inhibiting tumor growth. The efficacy results are shown in Figure 6A and Table 9. The mouse body weights were also monitored, and as shown in Figure 6B, no significant differences were observed in the mice's weights. Therefore, the TROP2-ISAC drug of the present invention demonstrated a significant tumor-suppressing effect, and the mice were in good general condition, indicating that side effects or off-target effects were minimal.

[0970] Table 9. Tumor inhibition rate on day 26

[0971] 6.2 Therapeutic Effects of hz1D9B10-Compound 5, hz1D9B10-Compound 8, and hz1D9B10-Compound 6 in the NCI-N87 Model

[0972] In this experiment, NCI-N87 cells were inoculated into CB17-SCID mice to determine the anti-tumor effect of the TROP2-ISAC antibody of the present invention.

[0973] CB17-SCID mice:

[0974] Female CB17-SCID mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., and were SPF-grade. The quality inspection unit was Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., and the certificate number was No. 110011221104924762. Upon arrival, the mice were quarantined for 3 days before the study began.

[0975] cell:

[0976] NCI-N87 cells were purchased from ATCC (CAT#:CRL-5822) and routine subculture was performed in strict accordance with the instructions for subsequent in vivo experiments. Cells were collected by centrifugation, resuspended in sterile PBS, and the cell density was adjusted to 10×10 6 On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal area of ​​CB17-SCID mice to establish the NCI-N87 tumor-bearing mouse model.

[0977] Dosage:

[0978] Eight days after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with tumor volumes of approximately 100-122 mm were selected. 3 The mice were divided into groups according to the tumor volume (6 mice per group). The dosage and method of administration are shown in Table 10. h-IgG (purchased from EQUITECH-BIO) was used as a negative control and administered on the 8th day after inoculation. The tumor volume and body weight of the mice were monitored twice a week. The body weight and tumor volume were measured before each administration. The relative tumor inhibition rate (TGI%) was calculated on the 25th day after inoculation. The calculation formula is as follows: TGI% = 100% * (tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume of the control group before administration). Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper. The tumor volume was calculated according to the following formula: V = L × W 2 / 2. Body weight was measured using an electronic balance.

[0979] Table 10. Experimental design

[0980] The tumor inhibition rates for the hz1D9B10-Compound 8, hz1D9B10-Compound 6, and hz1D9B10-Compound 5 groups were 92.67%, 116.78%, and 112.73%, respectively. The efficacy results are shown in Figure 6C and Table 11. We also monitored the weight of the mice, and as shown in Figure 6D, no significant differences were observed in their weights. Therefore, the TROP2-ISAC drug of the present invention demonstrated a significant inhibitory effect on tumors, and the mice were in good general condition, indicating that side effects or off-target effects were minimal.

[0981] Table 11. Tumor inhibition rate on day 25

[0982] Example 7 Therapeutic Effects of TROP2-ISAC in the BxPC3 Model

[0983] In this experiment, BxPC3 cells were inoculated into CB17-SCID mice to determine the anti-tumor effect of the TROP2-ISAC antibody of the present invention.

[0984] CB17-SCID mice:

[0985] Female CB17-SCID mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (SPF grade). The quality inspection unit is Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., and the certificate number is No. 110011221110560000. Mice were acclimated for 3 days after arrival before the study began.

[0986] cell:

[0987] BxPC3 cells were purchased from the Chinese Academy of Sciences Cell Bank (CAT#: TCHu012) and routine subculture was performed in strict accordance with the instructions for subsequent in vivo experiments. Cells were collected by centrifugation, resuspended in sterile PBS, and the cell density was adjusted to 15 × 10 6 On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal region of CB17-SCID mice to establish a BxPC3 tumor-bearing mouse model.

[0988] Dosage:

[0989] Eleven days after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with tumor volumes of approximately 76-122 mm were selected. 3 The mice were divided into groups according to the tumor volume (7 mice per group). The dosage and method of administration are shown in Table 12. h-IgG (purchased from EQUITECH-BIO) was used as a negative control and administered on the 11th day after inoculation. The tumor volume and body weight of the mice were monitored twice a week. The body weight and tumor volume were measured before each administration. The relative tumor inhibition rate (TGI%) was calculated on the 35th day after inoculation. The calculation formula is as follows: TGI% = 100% * (tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume of the control group before administration). Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper. The tumor volume was calculated according to the following formula: V = L × W 2 / 2. Body weight was measured using an electronic balance.

[0990] Table 12. Experimental design

[0991] Tumor inhibition results are shown in Figure 7A and Table 13. On day 35 post-inoculation, the hz1D9B10-Compound 6 and hz1D9B10-Compound 9 groups exhibited tumor inhibition rates of 155% and 150%, respectively. The efficacy results are shown in Figure 7A and Table 13. Mouse body weights were also monitored, and as shown in Figure 7B, no significant differences were observed. Therefore, the TROP2-ISAC drug of the present invention demonstrated a significant tumor-inhibiting effect, and the mice were in good general condition, indicating minimal side effects or off-target effects.

[0992] Table 13. Tumor inhibition rate on day 35

[0993] Example 8 Toxicity Study of TROP2-ISAC in BALB / C Mice

[0994] 8.1 Toxicity Studies of hz1D9B10-Compound 7 and hz1D9B10-Compound 9

[0995] This experiment used BALB / C mice. The toxicity of the drug was determined by injecting TROP2-ISAC drug intravenously twice with an interval of 7 days and observing the changes in body temperature and death of the mice.

[0996] BALB / C mice:

[0997] Female BALB / C mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., with an SPF grade and a quality inspection unit of Beijing Weitonglihua Experimental Animal Technology Co., Ltd. The certificate number is 110011221105222536. The mice were purchased from Weitonglihua and acclimated for 3 days after arrival before the study began.

[0998] Dosage:

[0999] The mice were divided into groups and weighed according to the experimental design in Table 14. TROP2-ISAC was injected intravenously on days 0 and 7, respectively. The rectal temperature of the mice was measured before administration on day 7, and at 20 min, 40 min, and 60 min after administration.

[1000] Table 14. Experimental design

[1001] The rectal temperature results are shown in Figure 8A and Table 15. After the second administration on D7, the rectal temperature of mice in the TROP2-compound 7 group decreased to 29.88°C at 60 minutes, and the rectal temperature of mice in the TROP2-compound 9 group decreased to 31.18°C at 60 minutes. Therefore, administration of TROP2-compound 7 and TROP2-compound 9 at 7 days intervals had a toxic effect on BALB / C mice.

[1002] Table 15. Rectal temperature of mice before and after administration on day 7

[1003] 8.2 Toxicity Studies of hz1D9B10-Compound 5, hz1D9B10-Compound 8, and hz1D9B10-Compound 6

[1004] This experiment used BALB / C mice. The toxicity of the drug was determined by injecting TROP2-ISAC drug intravenously twice with an interval of 7 days and observing the changes in body temperature and death of the mice.

[1005] BALB / C mice:

[1006] Female BALB / C mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., with an SPF grade and a quality inspection unit of Beijing Weitonglihua Experimental Animal Technology Co., Ltd. The certificate number is 110011221105981185. The mice were purchased from Weitonglihua and acclimated for 3 days after arrival before the study began.

[1007] Dosage:

[1008] The mice were divided into groups and weighed according to the experimental design in Table 16. TROP2-ISAC was injected intravenously on days 0 and 7, respectively. The rectal temperature of the mice was measured before administration on day 7, and at 20 min, 40 min, and 60 min after administration.

[1009] Table 16. Experimental design

[1010] Rectal temperature results are shown in Figure 8B and Table 17. Following the second administration on D7, only the TROP2-Compound 5 group showed a decrease in rectal temperature to 35.9°C at 60 minutes; no significant decrease was observed in the other groups. Therefore, administration of TROP2-Compound 8 and TROP2-Compound 6 at 7-day intervals had no toxic effects on BALB / C mice.

[1011] Table 17. Rectal temperature of mice before and after administration on day 7

[1012] Example 9 Toxic Effects of TROP2-ISAC in hTROP2 KI Mice

[1013] This experiment used hTROP2 KI mice. TROP2-ISAC drugs were injected intravenously twice to observe the changes in body temperature and death of mice to determine the toxicity of the drug.

[1014] hTROP2 KI transgenic mice:

[1015] Female hTROP2 KI (B-hTROP2) mice were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd., with an SPF grade and a quality inspection unit of Suzhou Xishan Biotechnology Co., Ltd. The certificate number is 3207262221100150728. The mice were purchased from Biocytogen and acclimated for 3 days after arrival before the study began.

[1016] Dosage:

[1017] The mice were divided into groups and weighed according to the experimental design in Table 18. TROP2-ISAC was injected intravenously on days 0 and 7, respectively. The rectal temperature of the mice was measured before administration on day 7, as well as 20 min, 40 min, and 60 min after administration.

[1018] Table 18. Experimental design

[1019] The rectal temperature results are shown in Figure 9 and Table 19. After the second dose on D7, the rectal temperature of mice in the TROP2-compound 8 group decreased to 34.55°C at 60 minutes, and the rectal temperature of mice in the TROP2-compound 9 group decreased to 29.20°C at 60 minutes. There was no significant decrease in the rectal temperature in the TROP2-compound 6 group. Therefore, administration of 10 mg / kg of TROP2-compound 6 at 7 days intervals had no toxic effect on hTROP2 KI mice.

[1020] Table 19. Rectal temperature of mice on day 7

[1021] Example 10: Dose-finding study of hz1D9B10-Compound 6 (hereinafter referred to as Conjugate A) administered by repeated intravenous infusion to cynomolgus monkeys for 4 weeks

[1022] This trial was conducted at Zhaoyan (Suzhou) New Drug Research Center Co., Ltd. The objectives of the study were to evaluate the potential toxicity of conjugate A administered intravenously or subcutaneously every two weeks or once a week for four consecutive weeks (three times in total) to cynomolgus monkeys, identify the target organs of toxicity, and investigate the immunogenicity and in vivo exposure levels of conjugate A.

[1023] A total of 8 cynomolgus monkeys (4 per sex) were used in this study and randomly divided into 4 groups according to body weight by sex. Animals in Groups 1 and 2 were given 10 and 30 mg / kg of Conjugate A, respectively, once every 2 weeks by intravenous infusion for a total of 3 doses, with a dosing volume of 5 mL / kg, a dosing rate of approximately 10 mL / kg / h, and a dosing time of approximately 30 minutes. Animals in Group 3 were given 15 mg / kg of Conjugate A once a week by intravenous infusion for a total of 4 doses, with a dosing volume of 5 mL / kg, a dosing rate of approximately 10 mL / kg / h, and a dosing time of approximately 30 minutes. Animals in Group 4 were given 30 mg / kg of Conjugate A once every 2 weeks by subcutaneous injection for a total of 3 doses, with a dosing volume of 2 mL / kg. Surviving animals in Groups 1, 2, and 4 were autopsied 7 days after the last dose (D36), and surviving animals in Group 3 were autopsied 7 days after the last dose (D29).

[1024] During the trial, the animals were subjected to clinical observation, body weight, food intake, body temperature, ophthalmology, clinical pathology (blood cell count, coagulation function, blood biochemistry), immune cell phenotype, cytokines, circulating immune complex CIC, anti-drug antibodies, gross anatomy, organ weighing and histopathological examination. At the same time, the contents of ISAC (conjugate A), total anti-(total antibodies of conjugate A) and small molecules (payload, linker-payload, cys-linker-payload) from conjugate A were measured and toxicokinetic analysis was performed.

[1025] Under the conditions of this study, female animals in the 30 mg / kg intravenous infusion and subcutaneous injection groups were either near death or found dead. Female animals in the 15 mg / kg dose group (intravenous administration QW) showed weight loss. In addition, the animals in each group mainly showed decreased food intake, immune-related toxicity (WBC, Neut, Lymph, CD3 + 、CD3 + CD4 + 、CD3 + CD8 + 、CD20 + The drug showed a decrease followed by an increase or recovery, with increases in Mono, IgG, IgM, MCP-1, IP-10, and IL-6, kidney-related toxicity, changes in red blood cell-related parameters (continuous decrease in RBC and HGB), and abnormal coagulation function (pale gums / cheeks, gum bleeding, skin erythema, decreased PLT, and undetectable coagulation / biochemical parameters in dead / moribund animals; increased Retic in other animals). The highest non-severe toxic dose (HNSTD) was 15 mg / kg (intravenous infusion QW).

[1026] After the first administration, the peak drug concentrations of conjugate A and total antibody of conjugate A in the serum of animals in the low and high dose groups (C max ) and drug exposure (AUC last ) increases with the increase of the dosage. After the first dose of 30 mg / kg of conjugate A was administered subcutaneously, the bioavailability of conjugate A in male and female animals (AUC last，皮下组 AUC last，静脉组 ) were 69% and 98% respectively. The bioavailability of total antibody of conjugate A in male and female animals was 60% and 89% respectively. After repeated administration, the blood drug concentration decreased rapidly, possibly due to the production of anti-drug antibodies. The calculated parameters and ratios are for reference only. After the first administration, the peak drug concentration of conjugate A in the animal body (C max ) and drug exposure (AUC last ) is basically the same as the total antibody of conjugate A.

[1027] The toxicokinetic parameters of conjugate A and total antibody of conjugate A in each group of animals are as follows:

[1028] Table 20. Toxicokinetic parameters of conjugate A and total antibody of conjugate A

[1029] Note: AUC of 15 mg / kg last * Statistics are for 0-168h. For 10mg / kg and 30mg / kg, statistics are for 0-336h.

[1030] Example 11 Therapeutic Effects of TROP2-ISAC in the CT26-hTROP2 Model

[1031] In this experiment, BALB / C mice were inoculated with CT26-hTROP2 cells to determine the anti-tumor effect of the TROP2-ISAC of the present invention.

[1032] BALB / C mice:

[1033] Female BALB / C mice were purchased from Zhejiang Weitonglihua Laboratory Animal Technology Co., Ltd. (SPF grade). The quality inspection unit was Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., and the certificate number was No. 20230510Abzz0619000585. Mice were acclimated for 3 days after arrival before the study began.

[1034] cell:

[1035] CT26-hTROP2 cells were constructed by Innovent Biologics and routinely subcultured for subsequent in vivo experiments. Cells were collected by centrifugation and resuspended in sterile PBS to adjust the cell density to 5 × 10 6 On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal region of BALB / C mice to establish a CT26-hTROP2 tumor-bearing mouse model.

[1036] Dosage:

[1037] Eight days after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with a tumor volume of approximately 100 mm were selected. 3The mice were divided into groups according to the tumor volume (7 mice per group). The dosage and method of administration are shown in Table 21. h-IgG (purchased from EQUITECH-BIO) was used as a negative control and administered on days 8, 11, 15, and 18 after inoculation. The tumor volume and body weight of the mice were monitored twice a week. The body weight and tumor volume were measured before each administration. The relative tumor inhibition rate (TGI%) was calculated on the 22nd day after inoculation. The calculation formula is as follows: TGI% = 100% * (tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume of the control group before administration). Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper. The tumor volume was calculated according to the following formula: V = L × W 2 / 2. Body weight was measured using an electronic balance.

[1038] Table 21. Experimental design

[1039] As shown in Figure 10A and Table 22, hz1D9B10-Compound 6 demonstrated a 52% tumor inhibition rate on day 22 post-inoculation. Mouse weights were also monitored, with no significant differences observed, as shown in Figure 10B. Survival rates were also observed, as shown in Figure 10C, with hz1D9B10-Compound 6 causing no mortality in the mice. Therefore, the TROP2-ISAC drug of the present invention demonstrated a significant tumor inhibition effect, and the mice were in good general condition with no significant side effects.

[1040] Table 22. Tumor inhibition rate on day 22

[1041] Example 12 Therapeutic Effect of TROP2-ISAC Combined with TROP2-ADC in the BxPC3 Model

[1042] In this experiment, BxPC3 cells were inoculated into CB17-SCID mice to determine the anti-tumor effect of TROP2-ISAC combined with TROP2-ADC.

[1043] CB17-SCID mice:

[1044] Female CB17-SCID mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., and were SPF grade. The quality inspection unit was Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., and the certificate number was No. 110011230103807533. The mice were acclimated for 3 days after arrival before the study began.

[1045] cell:

[1046] BxPC3 cells were purchased from the Chinese Academy of Sciences Cell Bank (CAT#: TCHu012) and routine subcultured in strict accordance with the instructions for subsequent in vivo experiments. Cells were collected by centrifugation and resuspended in sterile PBS to adjust the cell density to 1.5 × 10 7 On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal region of CB17-SCID mice to establish a BxPC3 tumor-bearing mouse model.

[1047] Dosage:

[1048] Thirteen days after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with a tumor volume of approximately 100 mm were selected. 3 The mice were divided into groups according to the tumor volume (7 mice per group). The dosage and method of administration are shown in Table 23. The drugs were administered on the 13th day after inoculation, and the tumor volume and body weight of the mice were monitored twice a week. The body weight and tumor volume were measured before each administration. The relative tumor inhibition rate (TGI%) was calculated on the 59th day after inoculation. The calculation formula is as follows: TGI% = 100% * (tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume of the control group before administration). Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured with a vernier caliper. The tumor volume was calculated according to the following formula: V = L × W 2 / 2. Body weight was measured using an electronic balance.

[1049] Table 23. Experimental design

[1050] Efficacy results are shown in Figure 11A and Table 24. On day 59 post-inoculation, the tumor inhibition rates for hz1D9B10-Compound 6 / TROP2 ADC (1 mg / kg), TROP2 ADC (5 mg / kg), hz1D9B10-Compound 6 + TROP2 ADC (1 + 1 mg / kg), and hz1D9B10-Compound 6 + TROP2 ADC (1 + 5 mg / kg) were 37%, 19%, 44%, 86%, and 106%, respectively. The hz1D9B10-Compound 6 + TROP2 ADC combination group showed greater efficacy than the hz1D9B10-Compound 6 and TROP2 ADC alone groups. Mouse body weights were also measured, and as shown in Figure 11B, no significant differences were observed. Therefore, the combination of TROP2-ISAC and TROP2-ADC demonstrated a significant synergistic inhibitory effect against BxPC3 tumors without significant toxic side effects in mice.

[1051] Wherein TROP2-ADC is hRS7-NT3 prepared in Example 4.8 above.

[1052] Table 24. Tumor inhibition rate on day 59

[1053] Example 13 Therapeutic Effect of TROP2-ISAC Combined with TROP2-ADC in the LK-2 Model

[1054] In this experiment, LK-2 cells were inoculated into CB17-SCID mice to determine the anti-tumor effect of TROP2-ISAC combined with TROP2-ADC.

[1055] CB17-SCID mice:

[1056] Female CB17-SCID mice were purchased from Beijing Weitonglihua Co., Ltd., and were SPF grade. The quality inspection unit was Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., and the certificate number was No. 110011231104065475. The mice were acclimated for 3 days after arrival before the study began.

[1057] cell:

[1058] LK-2 cells were purchased from Nanjing Kebai (CAT#: CBP60105) and routine subculture was performed in strict accordance with the instructions for subsequent in vivo experiments. Cells were collected by centrifugation, resuspended in sterile PBS, and the cell density was adjusted to 1.5×10 7 On day 0, 0.2 mL of cell suspension was subcutaneously inoculated into the right abdominal region of CB17-SCID mice to establish an LK-2 tumor-bearing mouse model.

[1059] Dosage:

[1060] Nine days after tumor cell inoculation, the tumor volume of each mouse was measured and the mice with a tumor volume of about 120 mm were selected. 3 The mice were divided into groups according to the tumor volume (7 mice per group). The dosage and method of administration are shown in Table 25. h-IgG (purchased from EQUITECH-BIO) was used as a negative control and administered on the 9th, 19th and 29th days after inoculation. The tumor volume and body weight of the mice were monitored twice a week. The body weight and tumor volume were measured before each administration. The relative tumor inhibition rate (TGI%) was calculated on the 36th day after inoculation. The calculation formula is as follows: TGI% = 100% * (tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume of the control group before administration). Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured with a vernier caliper. The tumor volume was calculated according to the following formula: V = L × W 2 / 2. Body weight was measured using an electronic balance.

[1061] Table 25. Experimental design

[1062] Efficacy results are shown in Figure 12A and Table 26. On day 36 after inoculation, the tumor inhibition rates of hz1D9B10-Compound 6, TROP2 ADC, and hz1D9B10-Compound 6 + TROP2 ADC were 29%, 17%, and 63%, respectively. The combination group showed greater efficacy than the single-drug groups. Mouse body weights were also measured, and as shown in Figure 12B, no significant differences were observed. Therefore, the combination of TROP2-ISAC and TROP2-ADC demonstrated a significant synergistic inhibitory effect against LK-2 tumors without significant toxic side effects in mice.

[1063] Wherein TROP2-ADC is hRS7-NT3 prepared in Example 4.8 above.

[1064] Table 26. Tumor inhibition rate on day 36

[1065] Example 14 High-dose toxicity study of TROP2-ISAC in BALB / C mice

[1066] This experiment used BALB / C mice. The toxicity of the drug was determined by injecting TROP2-ISAC drug intravenously twice with an interval of 7 days and observing the changes in body temperature and death of the mice.

[1067] BALB / C mice:

[1068] Female BALB / C mice were purchased from Beijing Weitonglihua Co., Ltd., of SPF grade, and the quality inspection unit was Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., with the certificate number 110011221111661541. The mice were acclimated for 3 days after arrival before the study began.

[1069] Dosage:

[1070] The mice were divided into groups and weighed according to the experimental design in Table 27, with 5 mice in each group. h-IgG (purchased from EQUITECH-BIO) was used as a negative control. TROP2-ISAC was intravenously injected on days 0 and 7, respectively. The rectal temperature of the mice was measured before administration on day 7 and 30 minutes and 60 minutes after administration.

[1071] Table 27. Experimental design

[1072] Rectal temperature results are shown in Figure 13A and Table 28. After dosing on day 7, rectal temperatures at 5 mg / kg, 15 mg / kg, and 45 mg / kg doses of hz1D9B10-Compound 6 did not decrease compared to pre-dose levels. Survival rates were also analyzed, and as shown in Figure 13B, no mice died at different doses of hz1D9B10-Compound 6. Therefore, high-dose administration of hz1D9B10-Compound 6 at 7-day intervals was not toxic to BALB / C mice.

[1073] Table 28. Rectal temperature of mice before and after administration on day 7

[1074] Example 15: Display library preparation of anti-FRα antibodies

[1075] Experimental strategy: Based on phage display technology, The scFv library was panned with human FOLR1 (FRα) for two rounds, and then the panned library was extracted from plasmids. The library fragments were transferred to a yeast system via PCR to further obtain highly functional molecules.

[1076] 15.1 Phage Display Antibody Panning

[1077] Removal of frozen human The scFv library recombinant phage was dissolved at room temperature, and the library was synthesized into 6 sub-libraries according to the different types of IgM / IgG antibodies displayed. PBST (PBS + 0.05% Tween 20) was added with 4% BSA and filtered and sterilized to prepare the blocking solution. 2x10 12 The recombinant phage was blocked at room temperature for 1 hour, and the Dynabeads were removed. TMBlock the M-280 (Invitrogen) with shaking at room temperature for 1 hour. Blocked magnetic beads were added to the blocked recombinant phage and shaken at room temperature for 1 hour to achieve negative selection. The centrifuge tube was placed on a magnetic rack to absorb the beads. The recombinant phage was aspirated and transferred to a new centrifuge tube. 30 pmol of biotinylated Human FOLR1 (Acro Biosystems) was added and allowed to bind at room temperature for 1.5 hours. The blocked magnetic beads were added to the antigen-recombinant phage complex for adsorption and shaking at room temperature for 0.5 hour. The beads were washed eight times with PBST buffer, collected, and washed with 0.5 ml of glycine elution buffer for 10 minutes at room temperature. The beads were placed on a magnetic rack to absorb the beads. The supernatant was removed and Tris was added for pH neutralization. After infection with TG1 competent cells, 100 μl of the diluted aliquot was plated for library size determination. Excess medium was removed by centrifugation, and the aliquot was plated onto a large 2xYT (2% glucose) plate and incubated overnight at 30°C for library scraping and recombinant phage preparation.

[1078] 15.2 Recombinant Phage Preparation

[1079] Scrape the output from the first round of screening from the culture plate and transfer a portion of the culture medium to a 2xTY (2% glucose) liquid culture plate to an OD600 of 0.1. Incubate at 37°C, 220 rpm, and reduce the OD600 to 0.5. Add an appropriate amount of helper phage M13K07 (PROGEN) and infect for 1 hour. Collect the cells by centrifugation and resuspend in 2xTY (2% glucose) medium. Incubate at 30°C overnight. Centrifuge the overnight culture to remove the precipitate. Add 1 / 4 volume of PEG / NaCl to the supernatant, incubate on ice for 1 hour, centrifuge for 20 minutes, remove the supernatant, retain the precipitate, resuspend in PBS, and assay the phage titer to complete the recombinant phage preparation.

[1080] Using human FOLR1 for two rounds of phage panning, the library was enriched to approximately 10 9 570 monoclonal clones were selected for ELISA to verify the binding ability of human FOLR1 and cyno FOLR1. The results showed that the cross binder reached 48%, and the next step of phage to yeast was possible. 8 E. coli for plasmid extraction, NanoDrop TM The concentration of the plasmid was determined at 2000 and the plasmid was frozen and stored for subsequent yeast library construction.

[1081] 15.3 Yeast Display Library Construction

[1082] Based on human A primer mix was designed based on the scFv library antibody sequence. Using plasmids extracted from the phage display library as templates, scFv fragments displaying the IgM and IgG antibody libraries were amplified. Using the yeast display plasmid pYDC011 as a template, upstream and downstream homology arms approximately 150 bp in length were amplified. Overlap PCR was used to amplify the full-length fragment, approximately 1200 bp in length. Plasmid pYDC011 was treated with the restriction endonuclease BamHI to linearize the vector. The full-length fragment and linearized vector were co-transformed into Saccharomyces cerevisiae EBY100 (purchased from ATCC) using an electroporator (BioRad). Transformed yeast were then transferred to the corresponding liquid deficient medium for overnight culture. Library size was determined by serial dilution and plated onto deficient medium plates. Library quality was then confirmed by sequencing. Successful transformations were screened overnight for library screening.

[1083] 15.4 Yeast Display Library Induction

[1084] The yeast library cultured overnight was centrifuged to collect the cells, resuspended and centrifuged twice using pre-cooled PBSA (PBS + 0.1% BSA) buffer, and the collected cells were transferred to an appropriate volume of YPGP induction medium (2% galactose, 2% peptone, 1% yeast extract, 0.54% Na2HPO4, 0.86% NaH2PO4.H2O) and induced overnight at 20°C for 72 hours to display the library scFv antibody fragments on the yeast surface for subsequent library staining screening.

[1085] 15.5 Yeast Display Library Screening

[1086] Yeast library was sorted using flow cytometry (BD): about 1x10 8 The yeast cells were washed three times with PBSA buffer and incubated with biotinylated human FOLR1 antigen and Mouse anti-Flag M2 (Sigma) at room temperature for 1 hour. The culture medium was discarded, the cells were eluted twice with PBSA buffer, mixed with SA-PE (Thermo Fisher) and Goat Anti Mouse-647 (Thermo Fisher) reagents, and incubated at room temperature for 20 minutes. The cells were eluted twice with pre-cooled PBSA buffer and resuspended in an appropriate volume of PBSA buffer. The cells were passed through a 40 μm mesh and transferred to a flow cytometry tube. The cells were detected using a BD FACSAria TM The positive bacterial colonies were circled, the positive yeast cells were sorted and transferred to liquid defective medium for overnight culture.

[1087] 15.6 Clone Identification for Yeast Display Library Screening

[1088] The bacterial colonies enriched three times by FACS sorting were plated on defective culture plates and cultured at 30°C for 72 hours until single colonies emerged. An appropriate number of colonies were selected and inoculated into liquid defective culture medium for activation and amplification. A portion of the culture medium was used for sequencing, and the remaining culture medium was centrifuged and the supernatant discarded. The cells were then inducible with YPGP for 72 hours. The cells were then incubated with a low concentration of biotinylated human FOLR1 antigen and Mouse Anti-Flag M2 (Sigma) with shaking at room temperature for 1 hour. The culture medium was discarded, and the cells were washed twice with PBSA buffer and mixed with SA-PE (Thermo Fisher) and Goat Anti-Mouse-647 (Thermo Fisher) reagents and incubated with shaking at room temperature for 20 minutes. The cells were washed twice with pre-chilled PBSA buffer and resuspended in an appropriate volume of PBSA buffer. The cells were passed through a 40 μm sieve and transferred to flow cytometer tubes for single-clone signal analysis using FACS. The PE signal represents the intensity of binding to the human FOLR1 antigen.

[1089] 15.7 Yeast Sequencing and Protein Expression

[1090] Yeast monoclones with high signal were selected and sent to GENEWIZ for sequencing and subsequent sequence analysis. The DNA sequence of the potential molecule was amplified into the eukaryotic expression vector pcDNA3.1 and ligated to the gene encoding the light chain constant region and the IgG1 heavy chain constant region. The successfully constructed plasmid was transfected into eukaryotic cells for antibody expression, yielding the full-length antibody BC1254-A1.

[1091] Example 16. Cysteine ​​mutation modification and expression of anti-FRα antibody

[1092] In order to obtain a more stable and potent immunoconjugate molecule, based on the cysteine ​​mutation modification of BC1254-A1 in Example 15, the two light chain constant regions of the BC1254-A1 antibody were modified to carry lambda light chain constant regions in which the amino acids at positions 160 and 166 (both according to EU numbering) were mutated to cysteine.

[1093] The main sequences of antibody BC1254-A1 and the modified antibody BC1254-A1-V2 are as follows:

[1094] Antibody sequence information:

[1095] 16.1 Plasmid construction: The sequence was synthesized by GeneWeiZ and loaded into the pTT5 vector.

[1096] 16.2 Protein Expression: Proteins were produced using the ExpiCHO™ Expression system (Gibco, A29133). Specifically, ExpiCHO-STM cells (Gibco) were passaged according to the desired transfection volume. The cell density was adjusted to 3.5 × 10 cells per day before transfection. 6 cells / ml. On the day of transfection, the cell density was adjusted to 6×10 6 Take a 50ml centrifuge tube and add transfection buffer reagent OptiPROTM SFM (Gibco, 12309019) at 8% of the transfection cell volume. Calculate the total amount of plasmid required for transfection based on 0.8μg / ml of cells, where the light chain: heavy chain plasmid mass ratio is 1:1. Use a 0.22μm filter membrane to filter the transfection buffer containing DNA plasmid into another new 50ml centrifuge tube. Add ExpiFectamineTM CHO reagent (Gibco, 100033022) to the filtered mixture at a DNA: Reagent ratio of 1:4. Mix thoroughly. After mixing the transfection reagent and plasmid DNA, immediately add it slowly to the cells, gently shaking the shaker while adding. Control the incubation time of the transfection reagent and plasmid to not exceed 5min; incubate at 37℃ with a shaker at 8% After 18-22 hours, Enhancer (Gibco, 100033019) was added at a volume of 6 μL / ml of cells, and Feed (Gibco, A29101-01) was added at a volume of 300 μL / ml of cells. The cells were cultured at 37°C, 120 rpm, and 8% CO2. The cell feed was collected on day 7 or when cell viability was ≤ 60%. The cell feed was mixed with diatomaceous earth (Sartorius, Cat 1000037025) (40 g of diatomaceous earth per 1 L of cell feed) and filtered using a 0.22 μm disposable vacuum filter. The supernatant was used for subsequent affinity purification.

[1097] 16.3 Purification of target protein by affinity chromatography: A HiTrap MabSelect PrismA (GE Healthcare, Cat#17549853) affinity chromatography column was used for affinity capture. Before purification, 10-20 column volumes of 0.1 M NaOH were passed through the tubing and affinity chromatography column, and then the tubing and column were washed with 10-20 column volumes of distilled water. The packed column was equilibrated with 5 column volumes of 1× PBS (Gibco). The filtered cell feed was passed through the column, and the packed column was washed with 10 column volumes of 1× PBS to remove non-specific binding proteins. The packed column was rinsed with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5), the eluate was collected, the pH was adjusted to 6.0 with 2 M Tris, and the column was sterilized by filtration.

[1098] 16.4 Antibody In Vitro Reduction and Oxidation: Add an appropriate amount of GSH to the purified antibody and adjust the reaction pH to 8.0 with 2M Tris. Incubate at room temperature overnight. Replace the reaction solution with PBS and store at 4°C until needed.

[1099] 16.5 Antibody Purification by Ion Exchange Chromatography: A Mono S 5 / 50GL (GE Healthcare) ion exchange chromatography column was used and placed in an AKTApure system (GE Healthcare). The AKTApure system equipped with the Mono S 5 / 50GL ion exchange chromatography column was detoxified with 0.5 M NaOH for 2 hours, and then the system and column were rinsed with distilled water. The column was equilibrated with 5-10 column volumes of loading buffer (20 mM NaPO4, pH 6.6) until the conductivity and pH stabilized. The protein obtained by affinity chromatography was diluted 10-fold with loading buffer and then loaded. The column was re-equilibrated with 5 column volumes of loading buffer. A linear elution gradient of 0-40% elution buffer (20 mM NaPO4, 1 M NaCl, pH 6.6) was performed over 30 column volumes, and samples were collected based on UV absorbance.

[1100] The purity of the collected samples in each fraction was tested by size exclusion chromatography (SEC), and the samples in the fractions with a purity greater than 95% were combined based on the SEC results.

[1101] The purified antibody solution was centrifuged at 4500 rpm for 30 minutes using a 15 ml ultrafiltration centrifuge tube. The protein was diluted with PBS and centrifuged again at 4500 rpm for 30 minutes. This operation was repeated several times to replace the buffer. The antibodies after buffer replacement were combined and the antibody concentration was measured. The composition and content of the antibodies were further qualitatively and quantitatively analyzed using a combination of capillary electrophoresis (CE-SDS) and liquid chromatography-mass spectrometry (LC-MS).

[1102] Example 17 ForteBio determines the affinity of antibodies to antigens

[1103] The equilibrium dissociation constants (KD) of the anti-FRα antibodies of the present invention for binding to human, monkey, and mouse FRα were determined using thin-layer biofilm interferometry (ForteBio). ForteBio affinity determinations were performed according to existing methods (Estep, P et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013. 5(2): p. 270-8).

[1104] Briefly, an AHC (Sartorius, 18-5060) sensor was equilibrated offline in assay buffer for 30 minutes, followed by online monitoring for 60 seconds to establish a baseline. The purified antibodies obtained as described above were then loaded online onto the AHC sensor (ForteBio) for ForteBio affinity measurement. The sensor with the loaded antibody was then exposed to the antigen FRα, after which the sensor was transferred to assay buffer for off-rate measurement. KD values ​​were analyzed using ForteBio analysis software. Antigens were obtained from: human (Acro, Cat#FO1-H82E2), monkey (Acro, Cat#FO1-C52H8), mouse (Acro, Cat#FO1-M5225), and rat (Sino, Cat#81073-R08H).

[1105] The antibody affinity test results are shown in Table 29. The antibody BC1254-A1 has a high affinity for the antigen FRα, and the mutated antibody BC1254-A1 V2 maintains a comparable equilibrium dissociation constant KD for the antigen.

[1106] Table 29 Affinity constants (M) of antigen-antibody binding detected by ForteBio

[1107] Note: ND means no data, NB means no binding.

[1108] Mirvetuximab is a control antibody from ImmunoGen, a FRα ADC For antibodies, sequences and sources, see U.S. Patent Application Publication No. US20200362029A1 (huMov19 (or M9346A)).

[1109] FR57 is a control antibody from ImmunoGen. The sequence and source are available in U.S. Patent Application Publication No. US20200362029A1.

[1110] Example 18 Antibody-dependent cellular phagocytosis (ADCP) functional assay

[1111] FcγRIIa-H reporter cells (G9871, Promega) were used to detect ADCP (Antibody-dependent cell-mediated phagocytosis) activity of FRα antibodies. These cells are engineered Jurkat T cells. When FcγRIIa on the cell surface is bound and activated, luciferase is released into the experimental system through the downstream signal NFAT (Nuclear Factor of Activated T), thereby detecting the degree of cell activation.

[1112] The specific method is as follows: a 96-well white flat-bottom cell culture plate was used, and 6×10 target cells SKOV-3 (HTB-77, ATCC) were added to each well. 4 and 3×10 effector FcγRIIa-H cells 4 Then, the corresponding concentration of FRα monoclonal antibody was added and the cells were incubated at 37°C for 6 hours. The culture plate was then removed and Bio-Glo (G7940, Promega) was added. The wavelength was detected using a microplate reader (Spark Multimode Microplate Reader, Tecan).

[1113] The results are shown in FIG14 . The ADCP activity of clone BC1254-A1 is superior to that of mirvetuximab, and it is suitable for the preparation of ISAC (Immune-Stimulating Antibody Conjugate).

[1114] Example 19 Verification of the Macrophage Endocytosis Function of Antibodies

[1115] The ADCP activity of the FRα antibody was verified using induced differentiated human M1 macrophages. The macrophages and target cells were fluorescently labeled separately. When the two underwent ADCP with the help of the antibody, a double-positive cell population could be observed.

[1116] M1 macrophage differentiation was induced: Monocytes were isolated from fresh human peripheral blood mononuclear cells (PBMCs, TPCS) using a human monocyte enrichment kit (19058, STEMCELL) without CD16 depletion. 6 Resuspend in AIM at a density of 1000 ng / ml. Medium CTS (A3021002, Gibco) was placed in a cell culture flask and cultured at 37°C for 4 hours. The medium was then replaced with AIM Culture medium CTS (A3021002, Gibco) + 10% inactivated fetal bovine serum (FBS, SH30406.05, Hyclone) + 25ng / ml human MCSF1 (216-MC-500, R&D). For the next seven days, culture medium AIM was used every three days. Medium CTS (A3021002, Gibco) + 10% inactivated fetal bovine serum (FBS, SH30406.05, Hyclone) was replaced halfway once. After seven days, medium AIM was used. The medium was half-changed with Medium CTS (A3021002, Gibco) + 10% inactivated fetal bovine serum (FBS, SH30406.05, Hyclone) + 100 ng / ml IFNg (285-IF-100 / CF, R&D), and the macrophage endocytosis experiment was performed the next day.

[1117] Macrophage endocytosis assay: After differentiation, macrophages were digested with Accutase (A6964-500ML, Sigma) and the cell density was adjusted using RPMI-1640 medium (22400-071, Gibco) containing 10% fetal bovine serum (FBS, SH30406.05, Hyclone). 1×10 5 Target SKOV-3 cells (HTB-77, ATCC) were labeled with CFSE (C34554, INVITROGEN) at 37°C for 10 min, washed twice with RPMI-1640 medium (22400-071, Gibco) containing 10% fetal bovine serum (FBS, SH30406.05, Hyclone), and 2.5×10 cells were added to each well of a 96-well low-adhesion plate. 4 Target cells and macrophages were mixed with gradiently diluted FRα antibodies by pipetting and incubated at 37°C for 4 hours.

[1118] Flow cytometry: Macrophages were labeled with CD14 PE / Cy7 (557742, BD) and then subjected to flow cytometry. Cells in the PE / Cy7 and CFSE double-positive area were identified as the target cell population.

[1119] The results are shown in FIG15 , showing that BC1254-A1 antibody can induce ADCP in M1 macrophages in the presence of SKOV-3.

[1120] Example 20 Preparation of Conjugates (FRαISAC and FRαADC)

[1121] 20.1 Preparation of BC1254-A1 V2-Compound 6

[1122] The specific method is as follows:

[1123] (a) Antibody BC1254-A1 V2 was dissolved in histidine buffer (20 mM, pH 6.5, Sigma, Lot# K5376054201);

[1124] (b) Add a reducing agent solution (TCEP, Aldrich, Catalog Number 646547, dissolved in water) and allow the reaction mixture to react at room temperature for 2 hours.

[1125] (i) The optimal concentration of BC1254-A1 V2 antibody is 5-15 mg / mL;

[1126] (ii) The optimal molar ratio of TCEP / mAb is 10–20;

[1127] (iii) The optimal reaction temperature is 25°C;

[1128] (iv) The optimal pH value of the reaction is 6.0-8.0.

[1129] (c) After the reduction is completed, the reducing agent is removed by desalting, ultrafiltration or dialysis purification.

[1130] (d) Add oxidant dehydroascorbic acid (dhAA, Aldrich, Catalog Number 261556, dissolved in DMSO) (DMSO, Sigma, Catalog Number 276855) and oxidize in a water bath for 2-3 h, wherein

[1131] (i) The optimal concentration of BC1254-A1 V2 antibody is 5-15 mg / mL;

[1132] (ii) The optimal molar ratio of dhAA / mAb is 20–40;

[1133] (iii) The optimal reaction temperature is 20-37°C;

[1134] (iv) The optimal pH value of the reaction is 6.0-8.0.

[1135] (e) adding an excess of linker-toxin (compound 6, dissolved in DMSO) to react with the antibody in step (d), and the solution

[1136] The volume ratio of DMSO in the mixture was 10%, and the reaction ...

Claims

1. An anti-TROP2 antibody or an antigen-binding fragment thereof, the antibody comprising: (i) Three complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:7, and three complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO:8; or (ii) Three complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:12, and three complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO:

14.

2. An anti-TROP2 antibody or antigen-binding fragment thereof, comprising a first heavy-chain complementarity-determining region (HCDR1), a second heavy-chain complementarity-determining region (HCDR2), a third heavy-chain complementarity-determining region (HCDR3), a first light-chain complementarity-determining region (LCDR1), a second light-chain complementarity-determining region (LCDR2), and a third light-chain complementarity-determining region (LCDR3), wherein: The HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or are respectively composed of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:

6.

3. The anti-TROP2 antibody or an antigen-binding fragment thereof according to claim 1 or 2, which comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises an amino acid sequence having at least 90% to at most 100% sequence identity with the amino acid sequence selected from SEQ ID NO:7 or 12, or is composed of the sequence; or, comprises the amino acid sequence selected from SEQ ID NO:7 or 12, or is composed of the sequence.

4. The anti-TROP2 antibody or an antigen-binding fragment thereof according to any one of claims 1-3, which comprises a light chain variable region (VL), wherein the light chain variable region comprises an amino acid sequence having at least 90% to at most 100% sequence identity with the amino acid sequence selected from SEQ ID NO:8 or 14, or is composed of the sequence; or, comprises the amino acid sequence selected from SEQ ID NO:8 or 14, or is composed of the sequence.

5. The anti-TROP2 antibody or an antigen-binding fragment thereof according to claim 1 or 2, which comprises a heavy chain variable region and a light chain variable region, wherein (i) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:7 or is composed of the amino acid sequence, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:8 or is composed of the amino acid sequence; or (ii) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:12 or is composed of the amino acid sequence, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:14 or is composed of the amino acid sequence.

6. The anti-TROP2 antibody or an antigen-binding fragment thereof according to any one of claims 1-5, which comprises an Fc region.

7. The anti-TROP2 antibody or antigen-binding fragment thereof according to claim 6, wherein the Fc region is the Fc of human IgG, for example, the Fc of human IgG1, the Fc of human IgG2, the Fc of human IgG3 or the Fc of human IgG4.

8. The anti-TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1-7, which comprises a heavy chain, wherein the heavy chain (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 selected from SEQ ID NO:9 or 15 or consists of the amino acid sequence; or (ii) comprises the amino acid sequence selected from SEQ ID NO:9 or 15 or consists of the amino acid sequence.

9. The anti-TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1-8, which comprises a light chain, wherein the light chain (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 selected from SEQ ID NO:10 or 16 or consists of the amino acid sequence; or (ii) comprises the amino acid sequence selected from SEQ ID NO:10 or 16 or consists of the amino acid sequence.

10. The anti-TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1-9, which comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:9 or consists of the same, and the light chain comprises the amino acid sequence of SEQ ID NO:10 or consists of the same.

11. The anti-TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1-10, wherein the antibody is a monoclonal antibody.

12. The anti-TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1-11, wherein the antibody is a humanized antibody or a chimeric antibody.

13. The anti-TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1-12, wherein the antigen-binding fragment is an antibody fragment selected from the following: Fab, Fab’, Fab’-SH, Fv, single-chain antibody (such as scFv), (Fab’)2, single-domain antibody such as VHH, dAb (domain antibody), bivalent antibody or linear antibody.

14. A fusion protein comprising the anti-TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1-13 15. An immunoconjugate comprising the anti-TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1-13 and other substances, such as toxins, small molecule drugs, cytotoxic agents, apoptosis agents, chelating agents, immunomodulators, such as anti-inflammatory agents or immunosuppressants.

16. The immunoconjugate according to claim 15, which comprises an immunomodulator, such as a TLR agonist, such as a TLR7 / 8 agonist.

17. An isolated nucleic acid encoding the anti-TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1-13.

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

19. A host cell comprising the nucleic acid of claim 17 or the vector of claim 18, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells (such as 293 cells or CHO cells, such as CHO-K cells or HEK293 cells) or other cells suitable for preparing an antibody or antigen-binding fragment thereof.

20. A method for preparing an anti-TROP2 antibody or antigen-binding fragment thereof, the method comprising a) culturing the host cell of claim 19 under conditions suitable for expressing a nucleic acid encoding the anti-TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1-13, b) optionally isolating the antibody or antigen-binding fragment thereof, c) optionally, the method further comprises recovering the anti-TROP2 antibody or antigen-binding fragment thereof from the host cell, optionally, the antibody is purified, such as purified by Protein A.

21. An anti-FRα antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising: Three complementarity-determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:33, and three complementarity-determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:

34.

22. An anti-FRα antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising a first heavy-chain complementarity-determining region (HCDR1), a second heavy-chain complementarity-determining region (HCDR2), a third heavy-chain complementarity-determining region (HCDR3), and a first light-chain complementarity-determining region (LCDR1), a second light-chain complementarity-determining region (LCDR2) and a third light-chain complementarity-determining region (LCDR3), wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 or are respectively composed of the amino acid sequences shown in SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:

32.

23. The anti-FRα antibody or antigen-binding fragment thereof according to claim 21 or 22, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), and the heavy chain variable region 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 shown in SEQ ID NO: 33, or consists of said sequence; or comprises the amino acid sequence shown in SEQ ID NO: 33, or consists of said sequence.

24. The anti-FRα antibody or antigen-binding fragment thereof according to any one of claims 21-23, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL), and the light chain variable region 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 shown in SEQ ID NO: 34, or consists of said sequence; or comprises the amino acid sequence shown in SEQ ID NO: 34, or consists of said sequence.

25. An anti-FRα antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: a VH comprising the amino acid sequence shown in SEQ ID NO: 33 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto or consisting of said amino acid sequence, and / or a VL comprising the amino acid sequence shown in SEQ ID NO: 34 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto or consisting of said amino acid sequence.

26. The anti-FRα antibody or antigen-binding fragment thereof according to claim 21 or 22, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 33 or consists of said amino acid sequence, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 34 or consists of said amino acid sequence.

27. The anti-FRα antibody or antigen-binding fragment thereof according to any one of claims 21-26, wherein the antibody or antigen-binding fragment thereof comprises an Fc region, for example, the Fc region is from the Fc region of IgG1, IgG2, IgG3 or IgG4, for example, the Fc region of human IgG1, IgG2, IgG3 or IgG4.

28. The anti-FRα antibody or antigen-binding fragment thereof according to any one of claims 21-27, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region, and the heavy chain constant region is from the constant region of IgG1, IgG2, IgG3 or IgG4, for example, the constant region of human IgG1, IgG2, IgG3 or IgG4, for example, the IgG1 heavy chain constant region (i) comprises the amino acid sequence of SEQ ID NO: 46 or consists of said amino acid sequence; or (ii) 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 of SEQ ID NO:

46.

29. The anti-FRα antibody or antigen-binding fragment thereof according to any one of claims 21-28, wherein the antibody or antigen-binding fragment thereof comprises a light chain constant region, and the light chain constant region is a lambda or kappa light chain constant region, such as a human lambda or kappa light chain constant region. Preferably, the light chain constant region (i) comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of the amino acid sequences of SEQ ID NOs: 38-41 and 45 or consists of said amino acid sequence; or (ii) comprises any one of the amino acid sequences of SEQ ID NOs: 38-41 and 45 or consists of said amino acid sequence.

30. The anti-FRα antibody or antigen-binding fragment thereof according to any one of claims 21-29, wherein the antibody or antigen-binding fragment thereof comprises one or two lambda light chain constant regions, and has a cysteine substitution (LLC160C) at position 160 (EU numbering) of the lambda light chain constant region.

31. The anti-FRα antibody or antigen-binding fragment thereof according to claim 30, wherein the lambda light chain constant region comprising LLC160C comprises the amino acid sequence shown in SEQ ID NO: 39 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 39 and comprises the amino acid sequence VKAGVCTTTPS (SEQ ID NO: 42).

32. The anti-FRα antibody or antigen-binding fragment thereof according to any one of claims 21-29, wherein the antibody or antigen-binding fragment thereof comprises one or two lambda light chain constant regions, and has a cysteine substitution (LLC166C) at position 166 (EU numbering) of the lambda light chain constant region.

33. The anti-FRα antibody or antigen-binding fragment thereof according to claim 32, wherein the lambda light chain constant region comprising LLC166C comprises the amino acid sequence shown in SEQ ID NO: 40 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 40 and comprises the amino acid sequence TTTPSCQSNNK (SEQ ID NO: 43).

34. The anti-FRα antibody or antigen-binding fragment thereof according to any one of claims 21-29, wherein the antibody or antigen-binding fragment thereof comprises one or two Lambda light chain constant regions, and has cysteine substitutions (LLC160C & LLC 166C) at positions 160 and 166 (EU numbering) of the Lambda light chain constant region.

35. The anti-FRα antibody or antigen-binding fragment thereof according to claim 34, wherein the Lambda light chain constant region containing LLC160C and LLC166C comprises the amino acid sequence shown in SEQ ID NO:41 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:41 and comprises the amino acid sequence VKAGVCTTTPSCQSNNK (SEQ ID NO:44).

36. The anti-FRα antibody or antigen-binding fragment thereof according to any one of claims 21-35, wherein the antibody or antigen-binding fragment thereof comprises (i) one or two Lambda light chain constant regions having a cysteine mutation at position 160 (Eu numbering); and / or (ii) one or two Lambda light chain constant regions having a cysteine mutation at position 166 (Eu numbering).

37. The anti-FRα antibody or antigen-binding fragment thereof according to any one of claims 21-36, wherein the antibody or antigen-binding fragment thereof comprises two identical heavy chains and two identical light chains, wherein the heavy chain comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:35 or consists of the same, and / or the light chain comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:36 or consists of the same.

38. The anti-FRα antibody or antigen-binding fragment thereof according to any one of claims 21-36, wherein the antibody or antigen-binding fragment thereof comprises two identical heavy chains and two identical light chains, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:35 or consists of the same, and / or the light chain comprises the amino acid sequence shown in SEQ ID NO:36 or consists of the same.

39. The anti-FRα antibody or antigen-binding fragment thereof according to any one of claims 21-36, wherein the antibody or antigen-binding fragment thereof comprises two identical heavy chains and two identical light chains, wherein the heavy chain comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:35 or consists of the same, and / or The light chain 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:

37.

40. The anti-FRα antibody or antigen-binding fragment thereof according to any one of claims 21-36, wherein the antibody or antigen-binding fragment thereof comprises two identical heavy chains and two identical light chains, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 35, and / or the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:

37.

41. The anti-FRα antibody or antigen-binding fragment thereof according to any one of claims 21-40, wherein the antibody is a humanized antibody or a chimeric antibody.

42. A fusion protein comprising the anti-FRα antibody or antigen-binding fragment thereof according to any one of claims 21-41.

43. An immunoconjugate comprising the antibody or antigen-binding fragment thereof against FRα according to any one of claims 21-41 and other substances, such as toxins, small molecule drugs, cytotoxic agents, apoptotic agents, chelating agents, immunomodulators, such as anti-inflammatory agents or immunosuppressive agents.

44. The immunoconjugate according to claim 43, which comprises an immunomodulator, such as a TLR agonist, such as a TLR7 / 8 agonist.

45. An isolated nucleic acid encoding the anti-FRα antibody or antigen-binding fragment thereof according to any one of claims 21-41.

46. A vector comprising the nucleic acid of claim 42, preferably the vector is an expression vector.

47. A host cell comprising the nucleic acid of claim 45 or the vector of claim 46, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells (such as 293 cells or CHO cells, such as CHO-K cells or HEK293 cells) or other cells suitable for preparing an antibody or antigen-binding fragment thereof.

48. A method for preparing an anti-FRα antibody or antigen-binding fragment thereof, the method comprising (a) culturing the host cell of claim 47 under conditions suitable for expressing a nucleic acid encoding the anti-FRα antibody or antigen-binding fragment thereof according to any one of claims 21-41, (b) optionally isolating the antibody or antigen-binding fragment thereof, (c) optionally, the method further comprises recovering the anti-FRα antibody or antigen-binding fragment thereof from the host cell, optionally, the antibody is purified, such as by Protein A purification.

49. A compound selected from the following or a pharmaceutically acceptable salt or solvate thereof: (1) The compound of formula (D-1): Wherein: R1 is an unsubstituted or C1-C6 alkyl substituted with one or more substituents selected from the following: hydroxy, halogen, cyano, C3-C6 cycloalkyl, amino, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C1-C6 alkoxy and C3-C6 cycloalkoxy; R2 and R3 are independently selected from hydrogen, C1-C6 alkyl, hydroxy, halogen, cyano, amino, C1-C6 alkoxy, aryl C1-C6 alkoxy, and C1-C6 alkoxycarbonyl, wherein the aryl is optionally substituted with one or more groups selected from: C1-C6 alkyl, hydroxy, halogen, cyano, amino, and C1-C6 alkoxy; R4 is an unsubstituted or C1-C6 alkyl substituted with one or more substituents selected from: hydroxy, halogen, cyano, C3-C6 cycloalkyl, aryl C1-C6 alkyl, amino, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 alkoxy substituted with NHR5, and C1-C6 alkylcarbonyloxy substituted with NHR5; wherein the aryl is optionally substituted with one or more groups selected from: C1-C6 alkyl, hydroxy, halogen, cyano, amino, C1-C6 alkoxy, and C1-C6 alkylamino; R5 is selected from C1-C6 alkyl and C1-C6 alkylcarbonyl, wherein the C1-C6 alkyl and C1-C6 alkylcarbonyl are optionally substituted with one or more substituents selected from: hydroxy, halogen, amino, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C1-C6 alkoxy, and C3-C6 cycloalkoxy; (2) Compound of formula (D-2): wherein: X is absent or is -NH-(C=O)-, -(C=O)-NH-, or C1-C6 alkylene, wherein the C1-C6 alkylene is optionally substituted with one or more substituents selected from hydroxy or halogen and one or more of the carbon atoms may be replaced with a group selected from -NH- and -(C=O)-; X1 is selected from CH or N; X2 is selected from CH2 or N; X3 is NR8, wherein R8 is selected from H and C1-C6 alkyl, and the C1-C6 alkyl is optionally substituted with hydroxy, C1-C6 alkoxy, and C1-C6 alkoxy substituted with amino; X4 is CH2 or C(=O); R6 and R7 are independently selected from H and C1-C6 alkyl, and the C1-C6 alkyl is optionally substituted with one or more groups selected from hydroxy, halogen, cyano, C3-C6 cycloalkyl, amino, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, and C1-C6 alkoxy; and represents a single bond or a double bond; Compound of formula (D-3): wherein: R9 is selected from hydroxy, amino, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, and C1-C6 alkoxy; R 10 and R 11 are independently selected from hydrogen and C1-C8 alkyl, wherein the C1-C8 alkyl is optionally substituted with one or more substituents selected from hydroxy, halogen, C1-C6 alkoxy and NR 15 R 16 , wherein R 15 and R 16 are independently selected from H, C1-C6 acyl and hydroxy-substituted C1-C6 acyl; R 12 is absent or is selected from hydrogen and aryl C1-C6 alkyl, wherein the aryl is optionally substituted with one or more substituents selected from hydroxy, halogen, C1-C6 alkoxy and heterocyclic C1-C6 alkyl, and the heterocyclic group is optionally substituted with one or more substituents selected from C1-C6 alkyl, C1-C6 alkoxy-C1-C6 alkyl and C1-C6 acyl; R 13 selected from hydrogen, a halogen, a hydroxyl group or an oxo group (=O); R 14 selected from hydrogen, halogen, hydroxyl, C1-C6 alkyl or C1-C6 alkoxy; r is 0 or 1 or 2; Y is selected from C or NR 17 , wherein R 17 is selected from hydrogen or C1-C6 alkyl optionally substituted by aryl or heterocyclic group, and said aryl and heterocyclic group are optionally substituted by substituents selected from C1-C6 acyl, C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, hydroxy C1-C6 alkyl and NR 18 R 19 -C1-C6 alkyl, wherein R 18 and R 19 are independently selected from H, C1-C6 alkyl and C1-C6 acyl; and represents a single bond or a double bond. The compound of claim 49, which has the structure shown in formula (D-1’): wherein R2 and R3 are as defined in claim 21; and R is selected from H and wherein R1' and R2' are independently selected from hydrogen and C1-C6 alkyl, and p' is an integer selected from 1-6.

51. The compound of claim 49, having the structure shown in formula (D-2) and wherein R6 and R7 are selected from C1-C6 alkyl, preferably n-propyl. The compound of claim 49, which has the structure shown in formula (D-3’): wherein R4' represents a C3-C6 alkyl optionally substituted with one or more substituents selected from hydroxy, halogen, amino, and C1-C6 alkoxy, preferably n-pentyl; R5’ is selected from H, C1-C6 alkyl, C1-C6 alkoxy-C1-C6 alkyl, C1-C6 acyl; preferably selected from H, acetyl, methoxyethyl. The compound of claim 49, which has the structure shown in formula (D-3”): Wherein: Q is selected from OR6’ and NR7’R8’; wherein R6’ is selected from H and C1-C6 alkyl; R7’ and R8’ are independently selected from H, C1-C6 acyl, hydroxy-substituted C1-C6 acyl, preferably both R7’ and R8’ are H, or one of R7’ and R8’ is H and the other is acetyl or hydroxyacetyl. The compound of claim 49, which has the structure shown in formula (D-3'''): Wherein: R9’ is C1-C6 alkyl, preferably C3-C6 straight-chain alkyl, such as n-butyl; r is 0 or 1; X5 is selected from C1-C6 alkylene, such as -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-CH2-; B is selected from aryl or heterocyclic group, such as phenyl, tetrahydroisoquinolinyl and piperidinyl, and the aryl and heterocyclic group are optionally substituted by C1-C6 acyl, C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, hydroxy C1-C6 alkyl or NR’R”-C1-C6 alkyl, and the R’ and R” are independently selected from H, C1-C6 alkyl and C1-C6 acyl. The compound of claim 49, which has the structure shown in formula (D-1”): Wherein R1’ and R2’ are independently selected from hydrogen and C1-C6 alkyl, preferably both R1’ and R2’ are methyl, or R1’ is hydrogen and R2’ is 3,3-dimethyl-butyl.

56. A compound according to any one of claims 49 - 55, selected from 57. A compound of formula (II) or a pharmaceutically acceptable salt or solvate thereof: L-(D) m (II) Wherein D is a compound as defined in any one of claims 21-28 or a pharmaceutically acceptable salt or solvate thereof; L has the following structure: Z’-L 1- E-L2-L3- wherein L3 is connected to D; Z’ is selected from L1 is -(CH2) n -C(=O)- or -(CH2) n -(O(CH2)2) t (CH2) n -C(=O)-; E is absent or is a peptide residue containing 2-10 amino acids, wherein the amino acid residues are natural amino acid residues or unnatural amino acid residues and are optionally substituted by C1-C6 alkyl, and the C-terminus of the peptide residue is covalently connected to L2; L2 does not exist or L3 is absent or is -C(=O)-*, -C(=O)((CH2) n O) t (CH2) n -*, or -NH(CH2)-*, where * indicates that the terminus is covalently linked to D; n is an integer from 0 to 10, t is an integer from 1 to 10, m is an integer from 1 to 6.

58. The compound or a pharmaceutically acceptable salt or solvate thereof according to claim 57, wherein E has the structure shown by the following formula: -X1-X2- wherein X1 represents -(N(R 0 )C(R 0 )2C(=O)) s -, wherein R 0 each independently represents H or C1-C6 alkyl; s = 0-10, preferably 2, 4, 6 and 8; X2 does not exist or is selected from 59. The compound or a pharmaceutically acceptable salt or solvate thereof according to claim 58, wherein E is selected from where s = 0-8.

60. The compound or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 57 - 59, wherein L- has a structure selected from the following: The compound or a pharmaceutically acceptable salt or solvate thereof according to claim 57, wherein the compound is selected from:

62. An immunoconjugate having the formula (I): Ab-(L-(D) m ) p (I) or a pharmaceutically acceptable salt or solvate thereof, wherein: Ab is a specific antigen-binding protein or a fragment thereof; L is a linker; D is a TLR agonist, preferably a TLR7 / 8 agonist; m is an integer from 1 to 6; and p is an integer from 1 to 16, such as 2-10.

63. The immunoconjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 62, wherein D in formula (I) is a compound as defined in any one of claims 49-56 or a pharmaceutically acceptable salt or solvate thereof.

64. The immunoconjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 62 or 63, wherein L-(D) in formula (I) m is a compound of formula (II) according to any one of claims 57-61 or a pharmaceutically acceptable salt or solvate thereof.

65. The immunoconjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 64, wherein -L- has a structure selected from the following: Where the left side of the structure is connected to Ab and the right side is connected to D.

66. The immunoconjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 64, wherein the L-(D) m has a structure selected from the following:

67. The immunoconjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 62-66, which has a DAR value of 1-10 (such as 2-8).

68. The immunoconjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 62, which is Among them, q represents the DAR, for example 1 - 10, such as 2 - 8, such as 3, 4, 5, 6, or 7.

69. The immunoconjugate of any one of claims 62 - 68 or a pharmaceutically acceptable salt or solvate thereof, wherein the Ab specifically binds to TROP2.

70. The immunoconjugate of claim 69 or a pharmaceutically acceptable salt or solvate thereof, wherein the Ab is an anti - TROP2 antibody or an antigen - binding fragment thereof as described in any one of claims 1 - 13.

71. The immunoconjugate of claim 69 or 70 or a pharmaceutically acceptable salt or solvate thereof, which has a DAR value or an average DAR value of 1 - 10 (such as 2 - 8).

72. A pharmaceutical composition comprising an antibody or an antigen - binding fragment thereof as described in any one of claims 1 - 13, the fusion protein of claim 14, the immunoconjugate of claim 15 or 16, the nucleic acid of claim 17, the vector of claim 18, the host cell of claim 19, the compound of any one of claims 49 - 56, or the immunoconjugate of any one of claims 62 - 71 or a pharmaceutically acceptable salt or solvate thereof, and optionally one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small - molecule drugs, or immunomodulators, and optionally pharmaceutical excipients.

73. A drug combination comprising an antibody or an antigen - binding fragment thereof as described in any one of claims 1 - 13, the fusion protein of claim 14, the immunoconjugate of claim 15 or 16, the nucleic acid of claim 17, the vector of claim 18, the host cell of claim 19, the compound of any one of claims 49 - 56, or the immunoconjugate of any one of claims 62 - 71 or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of claim 72, and one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small - molecule drugs, or immunomodulators.

74. A method for preventing or treating a tumor in a subject, the method comprising administering to the subject an effective amount of an antibody or an antigen - binding fragment thereof as described in any one of claims 1 - 13, the fusion protein of claim 14, the immunoconjugate of claim 15 or 16, the nucleic acid of claim 17, the vector of claim 18, the host cell of claim 19, the compound of any one of claims 49 - 56, or the immunoconjugate of any one of claims 62 - 71 or a pharmaceutically acceptable salt or solvate thereof, the pharmaceutical composition of claim 72, or the drug combination of claim 73.

75. The method of claim 74, wherein the tumor is cancer, preferably, the cancer has an elevated level of (such as at the nucleic acid or protein level) TROP2, for example, compared to a healthy individual or healthy tissue adjacent to the patient's cancer tissue.

76. The method according to claim 75, wherein the cancer is selected from non-small cell lung cancer, triple-negative breast cancer, head and neck squamous cell carcinoma, urothelial carcinoma, ovarian cancer, endometrial cancer, gastric cancer, colorectal cancer, and cervical cancer.

77. The method according to any one of claims 74-76, wherein the method further comprises administering to the patient one or more therapies, such as treatment modalities and / or other therapeutic agents. Preferably, the treatment modality includes radiotherapy or surgery, or the therapeutic agent includes a chemotherapeutic agent, an angiogenesis inhibitor, a cytokine, a cytotoxic agent, other antibodies, small molecule drugs, or an immunomodulator.

78. Use of an antibody or an antigen-binding fragment thereof according to any one of claims 1-13, a fusion protein according to claim 14, an immunoconjugate according to claim 15 or 16, a nucleic acid according to claim 17, a vector according to claim 18, a host cell according to claim 19, a compound according to any one of claims 49-56, or an immunoconjugate according to any one of claims 62-71 or a pharmaceutically acceptable salt or solvate thereof, a pharmaceutical composition according to claim 72, or a pharmaceutical combination according to claim 73 in the preparation of a medicament for treating a tumor.

79. The use according to claim 78, wherein the tumor is a cancer. Preferably, the cancer has an elevated level of (e.g., at the nucleic acid or protein level) TROP2, e.g., compared to a healthy individual or to healthy tissue adjacent to the cancer tissue of the patient. More preferably, the cancer is selected from non-small cell lung cancer, triple-negative breast cancer, head and neck squamous cell carcinoma, urothelial carcinoma, ovarian cancer, endometrial cancer, gastric cancer, colorectal cancer, and cervical cancer.

80. An immunoconjugate according to any one of claims 62-68 or a pharmaceutically acceptable salt or solvate thereof, wherein the Ab specifically binds to FRα.

81. The immunoconjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 80, wherein the Ab is an anti-FRα antibody or an antigen-binding fragment thereof according to any one of claims 21-41.

82. The immunoconjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 80 or 81, which has a DAR value or an average DAR value of 1-10 (e.g., 2-8).

83. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 21-41, a fusion protein according to claim 42, an immunoconjugate according to claim 43 or 44, a nucleic acid according to claim 45, a vector according to claim 46, a host cell according to claim 47, a compound according to any one of claims 49-56, or an immunoconjugate according to any one of claims 62-68 and 80-82 or a pharmaceutically acceptable salt or solvate thereof, and optionally one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators, and optionally pharmaceutical excipients.

84. A pharmaceutical combination comprising an antibody or antigen-binding fragment thereof according to any one of claims 21 - 41, a fusion protein according to claim 42, an immunoconjugate according to claim 43 or 44, a nucleic acid according to claim 45, a vector according to claim 46, a host cell according to claim 47, a compound according to any one of claims 49 - 56 or an immunoconjugate according to any one of claims 62 - 68 and 80 - 82 or a pharmaceutically acceptable salt or solvate thereof or a pharmaceutical composition according to claim 83, and one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.

85. The pharmaceutical combination according to claim 84, wherein the other therapeutic agent is an antibody-drug conjugate targeting FRα, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate.

86. A method for preventing or treating a tumor in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 21 - 41, a fusion protein according to claim 42, an immunoconjugate according to claim 43 or 44, a nucleic acid according to claim 45, a vector according to claim 46, a host cell according to claim 47, a compound according to any one of claims 49 - 56 or an immunoconjugate according to any one of claims 62 - 68 and 80 - 82 or a pharmaceutically acceptable salt or solvate thereof, a pharmaceutical composition according to claim 83 or a pharmaceutical combination according to claim 84 or 85.

87. The method according to claim 86, wherein the tumor is cancer, preferably, the cancer has an elevated level of (e.g., at the nucleic acid or protein level) FRα, for example, compared to a healthy individual or healthy tissue adjacent to the cancer tissue of the patient.

88. The method according to claim 87, wherein the cancer is selected from non-small cell lung cancer, triple-negative breast cancer, head and neck squamous cell carcinoma, urothelial carcinoma, ovarian cancer, endometrial cancer, gastric cancer, colorectal cancer, cervical cancer.

89. The method according to any one of claims 86 - 88, wherein the method further comprises administering to the patient one or more therapies, such as treatment modalities and / or other therapeutic agents, preferably, the treatment modality includes radiotherapy or surgery, or the therapeutic agent includes chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.

90. The method according to claim 89, wherein the other therapeutic agent is an antibody-drug conjugate as defined in claim 85. Use of an antibody or antigen-binding fragment thereof according to any one of claims 21-41, a fusion protein according to claim 42, an immunoconjugate according to claim 43 or 44, a nucleic acid according to claim 45, a vector according to claim 46, a host cell according to claim 47, a compound according to any one of claims 49-56 or an immunoconjugate according to any one of claims 62-68 and 80-82 or a pharmaceutically acceptable salt or solvate thereof, a pharmaceutical composition according to claim 83 or a pharmaceutical combination according to claim 84 or 85 in the preparation of a medicament for treating a tumor.

92. Use according to claim 91, wherein the tumor is cancer, preferably, the cancer has an elevated level of (e.g., at the nucleic acid or protein level) FRα, e.g., compared to a healthy individual or healthy tissue adjacent to the cancer tissue of the patient, more preferably, the cancer is selected from non-small cell lung cancer, triple-negative breast cancer, head and neck squamous cell carcinoma, urothelial carcinoma, ovarian cancer, endometrial cancer, gastric cancer, colorectal cancer, cervical cancer.