Multispecific antigen-binding molecule and use thereof

Abstract

Provided are a multispecific antigen-binding molecule, a chimeric antigen receptor comprising same, a cell expressing the described chimeric antigen receptor, and use thereof. The multispecific antigen-binding molecule comprises a multispecific antibody. The multispecific antibody comprises: a first functional region targeting CD19, a second functional region targeting CD22, and a third functional region targeting BCMA, wherein the first functional region is an anti-CD19 heavy-chain antibody or an antigen-binding fragment thereof, the second functional region is an anti-CD22 heavy-chain antibody or an antigen-binding fragment thereof, and the third functional region is an anti-BCMA heavy-chain antibody or an antigen-binding fragment thereof. The described multispecific antibody has a ligand-binding ability and a cell-binding ability.

Description

Multispecific antigen-binding molecules and their applications Technical Field

[0001] This disclosure relates to the fields of biomedical or biopharmaceutical technology, and more specifically to a multispecific antigen-binding molecule and its applications. Background Technology

[0002] CAR (Chimeric Antigen Receptor) is a genetically engineered artificial receptor molecule that endows immune effector cells (such as T lymphocytes) with specificity for a target antigen epitope, thereby enhancing the T lymphocyte's ability to recognize antigen signals and activate, and subsequently reinfusing lymphocytes to achieve an anti-tumor effect. Several chimeric antigen receptor T-cell (CAR-T) drugs, represented by Kymriah and Yescarta, have been approved for marketing, marking a breakthrough in cell therapy for hematological malignancies and continuously bringing new hope to cancer patients. Studies have shown that multiple cell surface antigen targets have been discovered and applied to related CAR cell therapies, such as CD19 and BMCA, which have achieved great success in hematological malignancies, especially relapsed / refractory B-cell malignancies. However, CAR-T also exhibits drug resistance in clinical applications. For example, in patients with B-cell acute lymphoblastic leukemia (B-ALL) receiving CD19-targeted CAR-T cell therapy, up to 25% relapse, exhibiting CD19 negativity or low CD19 levels. This phenomenon is known as antigen escape. The main reason is that there is downregulation or loss of a single target, and the treatment effect is not as expected.

[0003] Multi-target CAR-T cells are primarily designed to target different tumor antigens, including dual CAR-T cells with two CARs or tandem CAR-T cells with multiple antibodies. Theoretically, this multi-target CAR-T approach can overcome tumor antigen heterogeneity and tumor antigen escape, a concept that has been validated clinically. Currently, the FDA has approved six CAR-T cell therapies; besides two targeting BCMA (Abecma and Carvykti), the other four are CD19 single-target CAR-T cells. Furthermore, CD19 is currently the most mature target in CAR-T development. Therefore, developing dual-target CAR-T cells based on CD19 CAR-T cells is the most common development route. The most common target combination is CD19 / CD22. CD22 is one of the inhibitory co-receptors on the surface of B cells and is expressed on the surface of most B-cell malignancies. The CD19 / CD22 target combination can cover various cell subpopulations in patients with primary and relapsed B-cell hematological malignancies. Meanwhile, even with CD19 loss, CD19 / CD22 CAR-T cells can still control the disease by binding to CD22, preventing disease progression and tumor recurrence; and the likelihood of tumor cells losing both CD19 and CD22 at the same time is very low.

[0004] Additionally, Gracell Biotech has an investigational CD19 / BCMACAR-T pipeline, GC012F. The NDMMIIT clinical trial showed an overall response rate (ORR) of 100% and a strict complete response rate (MRD-sCR) of 95.5% with negative minimal residual disease. Compared to traditional single-target therapies, the CD19 / BCMA dual-target design can better improve efficacy and reduce the probability of relapse caused by antigen escape.

[0005] In summary, the multi-target CAR-T strategy has shown therapeutic potential in hematological malignancies. Our aim is to leverage the advantages of nanobodies to construct dual-targeting or even triple-targeting CAR-Ts, thereby improving anti-tumor efficacy while ensuring safety. Summary of the Invention

[0006] This disclosure provides a multispecific antigen-binding molecule comprising a multispecific antibody or an antigen-binding fragment thereof, wherein the multispecific antibody contains: a first functional region targeting CD19, a second functional region targeting CD22, and a third functional region targeting BCMA;

[0007] The first functional region is an anti-CD19 heavy chain antibody or its antigen-binding fragment, the second functional region is an anti-CD22 heavy chain antibody or its antigen-binding fragment, and the third functional region is an anti-BCMA heavy chain antibody or its antigen-binding fragment.

[0008] This disclosure also provides a fusion protein comprising the multispecific antigen-binding molecule described herein and other peptides. In some embodiments, the other peptides are located at the N-terminus and / or C-terminus of the multispecific antigen-binding molecule. In some embodiments, the other peptides include peptides that localize the multispecific antigen-binding molecule to different organelles, tags for purification or for immune responses, transmembrane proteins or their transmembrane regions, chimeric antigen receptors or components thereof. In some embodiments, the fusion protein is a chimeric antigen receptor whose antigen-binding domain comprises the multispecific antigen-binding molecule. The chimeric antigen receptor comprises: an optional signal peptide sequence, an antigen-binding domain containing the multispecific antigen-binding molecule, a hinge region, a transmembrane region, and an intracellular region.

[0009] This disclosure also provides nucleic acid molecules having sequences selected from any of the following:

[0010] (1) The coding sequence of the multispecific antigen-binding molecule or fusion protein described in any of the embodiments herein;

[0011] (2)(1) complementary sequences.

[0012] This disclosure also provides a nucleic acid construct comprising the nucleic acid molecules described herein.

[0013] In some implementations, the nucleic acid construct is a cloning vector, expression vector, or integration vector.

[0014] This disclosure also provides phages comprising the multispecific antigen-binding molecules described in any embodiment herein.

[0015] In some embodiments, the multispecific antigen-binding molecule is displayed on the surface of the phage.

[0016] This disclosure also provides a host cell, selected from:

[0017] (1) Expressing and / or secreting the multispecific antigen-binding molecule or fusion protein as described in any of the embodiments herein;

[0018] (2) Contains the nucleic acid molecules described herein; and / or

[0019] (3) Includes the nucleic acid constructs described in this article.

[0020] In some embodiments, the host cell is an immune effector cell, preferably a T cell.

[0021] This disclosure also provides a method for generating a multispecific antigen-binding molecule or fusion protein according to any embodiment of the present invention, comprising:

[0022] The host cells described herein are cultured under conditions suitable for producing multispecific antigen-binding molecules or fusion proteins (e.g., nanobodies or their antigen-binding fragments, monovalent or multivalent nanobodies or single-domain antibodies, or multispecific nanobodies or single-domain antibodies), and optionally, the multispecific antigen-binding molecules or fusion proteins are purified from the culture.

[0023] Nucleic acid molecules encoding multispecific antigen-binding molecules or fusion proteins of any of the embodiments described herein are incubated under conditions suitable for the translation of DNA or RNA in a non-cellular system (e.g., solution).

[0024] This disclosure also provides a pharmaceutical composition comprising a multispecific antigen-binding molecule, fusion protein, nucleic acid molecule, nucleic acid construct, bacteriophage or host cell as described in any embodiment herein, and pharmaceutically acceptable excipients.

[0025] In some embodiments, the pharmaceutical composition is used to treat diseases or conditions associated with CD19, CD22, and / or BCMA expression, such as cancer.

[0026] This disclosure also provides the use of the multispecific antigen-binding molecules, fusion proteins, nucleic acid molecules, nucleic acid constructs, bacteriophages, or host cells described in any embodiment herein in the preparation of engineered immune cells (e.g., T cells).

[0027] This disclosure also provides the use of the multispecific antigen-binding molecules, fusion proteins, nucleic acid molecules, nucleic acid constructs, bacteriophages, or host cells described in any embodiment herein in the preparation of medicaments for the prevention or treatment of diseases or conditions associated with CD19, CD22, and / or BCMA expression.

[0028] In some implementations, the disease or condition is cancer, such as selected from B-cell acute lymphoblastic leukemia (“BALL”), T-cell acute lymphoblastic leukemia (“TALL”), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), multiple myeloma, follicular lymphoma, spleen, marginal zone lymphoma, mantle cell lymphoma, indolent B-cell lymphoma or Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, relapsed or refractory acute lymphoblastic leukemia (r / r ALL), relapsed or refractory diffuse large B-cell lymphoma (r / r DLBCL), and relapsed or refractory follicular lymphoma (r / r FL).

[0029] This disclosure also provides a method for treating or preventing diseases or conditions associated with CD19, CD22, and / or BCMA expression, the method comprising administering to a patient in need a therapeutically effective amount of a multispecific antigen-binding molecule, nucleic acid molecule, nucleic acid construct, or host cell as described in any embodiment of this disclosure, or a pharmaceutical composition as described in any embodiment of this disclosure. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of this disclosure, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of this disclosure and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1: Detection of binding of the first batch of candidate trispecific antibodies to Raji CD19 KO cells.

[0032] Figure 2: Detection of binding of the first batch of candidate trispecific antibodies to Raji CD22 KO cells.

[0033] Figure 3: Binding detection of the first batch of candidate trispecific antibodies with RPMI8226 cells.

[0034] Figure 4: Binding detection of the second batch of candidate trispecific antibodies with Raji WT cells.

[0035] Figure 5: Binding detection of the second batch of candidate trispecific antibodies with Raji CD19 KO cells.

[0036] Figure 6: Binding detection of the second batch of candidate trispecific antibodies with Raji CD22 KO cells.

[0037] Figure 7: Binding detection of the second batch of candidate trispecific antibodies with RPMI8226 cells.

[0038] Figure 8: Schematic diagram of the chimeric antigen receptor structure prepared by using trispecific antibodies as antigen-binding domains.

[0039] Figure 9: Positive rate of trispecific CAR-T prepared using trispecific antibodies as the antigen-binding domain of CAR.

[0040] Figure 10: Classification results of memory cell phenotypes of trispecific CAR-T cells.

[0041] Figure 11: Trispecific CAR-T cells and CD22 +The results of Raji cell co-culture and repeated stimulation of CAR-T cells are shown in Figure 11. A represents the tumor cell killing rate, B represents the change in CAR positivity rate, C represents the fold increase of CAR-T cells, and D represents the IFN-γ secretion results.

[0042] Figure 12: Trispecific CAR-T cells and CD19 + The results of Raji cell co-culture and repeated stimulation of CAR-T cells are shown in Figure 12. A represents the tumor cell killing rate, B represents the change in CAR positivity rate, C represents the fold increase of CAR-T cells, and D represents the IFN-γ secretion results.

[0043] Figure 13: Results of repeated stimulation of CAR-T cells with trispecific CAR-T cells and RPMI-8226 cells. In Figure 13, A represents the tumor cell killing rate, B represents the change in CAR positivity rate, C represents the fold increase of CAR-T cells, and D represents the IFN-γ secretion results.

[0044] Figure 14: Body weight changes in mice 49 days after a single injection of trispecific CAR-T cells.

[0045] Figure 15: Fluorescence detection results of tumor growth in mice after a single injection of high, medium, and low doses of trispecific CAR-T cells.

[0046] Figure 16: Comparison of fluorescence detection results of tumor growth in mice after a single injection of high doses of trispecific CAR-T and CD19 CAR-T.

[0047] Figure 17: Changes in the number of positive CAR-T cells in peripheral blood of mice after a single injection of high doses of trispecific CAR-T and CD19 CAR-T.

[0048] Figure 18: Killing rate of CAR-T cells prepared by 6h process and tumor cells after multiple rounds of co-culture.

[0049] Figure 19: Fission ratio of CAR-T cells prepared by the 6h process and tumor cells after multiple rounds of co-culture.

[0050] Figure 20: Changes over time in tumor volume, mouse body weight, number of CAR-T cells in peripheral blood, and proportion of positive CAR-T cells in Raji tumor mouse model after 6 hours of injection.

[0051] Figure 21: Fluorescence detection results of tumor growth in Raji tumor mouse model after injection of CAR-T cells prepared by the 6h process.

[0052] Figure 22: PET-CT images of representative CR patients in the LD cohort at 9 months.

[0053] Figure 23: Changes in the number of CAR-T cells in peripheral blood of the LD cohort over time.

[0054] Figure 24: Changes in the number of CAR-T cells in peripheral blood of the NLD cohort over time. Detailed Implementation

[0055] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are all standard procedures widely used in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0056] Through extensive and in-depth research and screening, the authors of this publication have discovered a class of anti-CD19, anti-CD22, and anti-BCMA nanobodies and their antigen-binding fragments, which can specifically recognize CD19, CD22, or BCMA and bind with high affinity.

[0057] The inventors have creatively fused different humanized nanobodies into new antibodies through protein recombination, obtaining humanized trispecific antibodies that can bind to CD19, CD22, and BCMA. These antibodies can effectively bind to cells expressing CD19, CD22, and / or BCMA, exhibit good functional activity, and show no tissue cross-reactivity.

[0058] This disclosure provides a multispecific antigen-binding molecule comprising a multispecific antibody or an antigen-binding fragment thereof. The multispecific antibody contains a first functional region targeting CD19 and a second functional region targeting CD22. The first functional region is an anti-CD19 heavy chain antibody or an antigen-binding fragment thereof, and the second functional region is an anti-CD22 heavy chain antibody or an antigen-binding fragment thereof. The first and second functional regions are independently comprised of one, two, or more, preferably one of each. The connection order of the first and second functional regions is not particularly limited. In some embodiments, the bifunctional antibody has the following structure from the N-terminus to the C-terminus: a first functional region targeting CD19, an optional linker, and a second functional region targeting CD22. In some embodiments, the bifunctional antibody has the following structure from the N-terminus to the C-terminus: a second functional region targeting CD22, an optional linker, and a first functional region targeting CD19.

[0059] The multispecific antibody further comprises a third functional region targeting BCMA, wherein the third functional region is an anti-BCMA heavy chain antibody or its antigen-binding fragment. The first, second, and third functional regions are each independently represented by one, two, or more, preferably one of each.

[0060] The connection order of the first, second, and third functional regions is not particularly limited. In some embodiments, the bifunctional antibody has the following structure from N-terminus to C-terminus or from C-terminus to N-terminus: a first functional region targeting CD19, an optional adapter, a second functional region targeting CD22, an optional adapter, and a third functional region targeting BCMA. In some embodiments, the bifunctional antibody has the following structure from N-terminus to C-terminus or from C-terminus to N-terminus: a second functional region targeting CD22, an optional adapter, a first functional region targeting CD19, an optional adapter, and a third functional region targeting BCMA. In some embodiments, the bifunctional antibody has the following structure from N-terminus to C-terminus or from C-terminus to N-terminus: a second functional region targeting CD22, an optional adapter, a third functional region targeting BCMA, an optional adapter, and a first functional region targeting CD19.

[0061] The first, second, and third functional regions can be directly connected or connected via a linker. The linker sequence is not limited and can be any amino acid linker sequence known in the art suitable for heavy chain antibodies or their antigen-binding fragments. In some embodiments, the linker is a linker composed of multiple Gs and Ss, such as (GGSGG)p or (G4S)m, wherein m, n, and p are each independently a positive integer from 1 to 10 (preferably 1 to 6, more preferably 1 to 4). In some embodiments, the linker is a linker composed of multiple As, such as AAA. In some embodiments, the linker is a linker composed of multiple Gs, Ss, and As.

[0062] As used herein, when referring to the amino acid sequence of the CD19 protein, it includes the full-length CD19 protein, or an extracellular fragment of CD19 or its fusion protein. However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of the CD19 protein without affecting its biological function. Therefore, in this disclosure, the term "CD19 protein" should include all such sequences, including its natural or artificial variants. Furthermore, when describing a sequence fragment of the CD19 protein, it also includes the corresponding sequence fragment from its natural or artificial variants.

[0063] As used herein, when referring to the amino acid sequence of the CD22 protein, it includes the full-length CD22 protein, or an extracellular fragment of CD22 or a fusion protein thereof. However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of the CD22 protein without affecting its biological function. Therefore, in this disclosure, the term "CD22 protein" should include all such sequences, including its natural or artificial variants. Furthermore, when describing a sequence fragment of the CD22 protein, it also includes the corresponding sequence fragment from its natural or artificial variants.

[0064] As used herein, when referring to the amino acid sequence of a BCMA protein, it includes the full-length BCMA protein, or an extracellular fragment of BCMA or its fusion protein. However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of a BCMA protein without affecting its biological function. Therefore, in this disclosure, the term "BCMA protein" should include all such sequences, including its natural or artificial variants. Furthermore, when describing a sequence fragment of a BCMA protein, it also includes the corresponding sequence fragment from its natural or artificial variants.

[0065] As used in this article, the term EC50 refers to the half-maximal effect concentration, which is the concentration that produces 50% of the maximum effect.

[0066] In this document, the term "antibody" includes monoclonal antibodies, antibody compositions and single-chain molecules with multi-epitope specificity, and antibody fragments, especially antigen-binding fragments, such as Fab, F(ab')2, and Fv. In this document, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.

[0067] The "heavy chain antibody" described in this article refers to antibodies derived from camelids or cartilaginous fishes. Compared to the aforementioned four-chain antibodies, heavy chain antibodies lack the light chain and heavy chain constant region 1 (CH1), containing only two heavy chains composed of a variable region (VHH) and other constant regions. The variable region is connected to the constant region via a hinge-like structure. Each heavy chain of camelid heavy chain antibodies contains one variable region (VHH) and two constant regions (CH2 and CH3), while each heavy chain of cartilaginous fish heavy chain antibodies contains one variable region and five constant regions (CH1-CH5). The antigen-binding fragment of heavy chain antibodies includes VHH or single-chain heavy chain antibodies. By fusing with the constant region of human IgG Fc, heavy chain antibodies can possess the CH2 and CH3 regions of human IgG Fc.

[0068] As used herein, the terms "nanobody," "single-domain antibody," "heavy chain variable region domain of a heavy chain antibody," and "VHH" are used interchangeably to refer to the VHH that specifically recognizes and binds to the antigen. The VHH is the variable region of a heavy chain antibody. Typically, a VHH contains three CDRs and four FRs. Preferably, in this disclosure, the complementarity-determining region (CDR) of the heavy chain antibody or VHH against CD19 includes: the CDR1 sequence shown in any one of SEQ ID NO: 1-5, the CDR2 sequence shown in any one of SEQ ID NO: 6-10, and the CDR3 sequence shown in any one of SEQ ID NO: 11-15; the CDR1 sequence of the heavy chain antibody or VHH against CD22 includes: the CDR1 sequence shown in any one of SEQ ID NO: 16-22, the CDR2 sequence shown in any one of SEQ ID NO: 23-29, and the CDR3 sequence shown in any one of SEQ ID NO: 30-36; the CDR1 sequence of the heavy chain antibody or VHH against BCMA includes: the CDR1 sequence shown in any one of SEQ ID NO: 37-40, the CDR2 sequence shown in any one of SEQ ID NO: 41-44, and the CDR3 sequence shown in any one of SEQ ID NO: 45-48.

[0069] In some embodiments, the complementarity-determining region of the anti-CD19 heavy chain antibody comprises: CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:1, 6, and 11, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:2, 7, and 12, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:3, 8, and 13, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:4, 9, and 14, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:5, 10, and 15, respectively.

[0070] The complementarity-determining region of the anti-CD22 heavy chain antibody comprises: CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:16, 23, and 30, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:17, 24, and 31, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:18, 25, and 32, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:19, 26, and 33, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:20, 27, and 34, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:21, 28, and 35, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:22, 29, and 36, respectively.

[0071] The complementarity-determining region of the anti-BCMA heavy chain antibody includes: CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:37, 41, and 45, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:38, 42, and 46, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:39, 43, and 47, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:40, 44, and 48, respectively.

[0072] In some embodiments, the multispecific antibody further comprises: a first functional region targeting CD19, a second functional region targeting CD22, and a third functional region targeting BCMA. The sequence of functional regions and the complementarity-determining region sequence in the multispecific antibody are shown in Table 1.

[0073] Table 1: Functional region linkage sequence and complementarity-determining region sequence in multispecific antibodies

[0074] Single-domain antibodies are the smallest functional antigen-binding fragments. Typically, antibodies that are naturally missing the light chain and heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region.

[0075] The term "variable" refers to the wide variation in certain segments within a variable domain within the antibody sequence. A variable domain (or variable region) contains an antigen-binding site, mediating antigen binding and defining the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across all amino acids spanned by the variable domain. For heavy chain antibodies or VHHs, the variable regions include CDR1, CDR2, and CDR3. The more highly conserved portions of the variable domain are called backbone regions (FRs). The variable domains of native heavy chains (and light chains) each contain four FR regions (FR1, FR2, FR3, and FR4), which mostly adopt a β-sheet conformation and are linked by three HVRs that form a ring connection and, in some cases, part of the β-sheet structure. The CDRs in each chain are held together very closely by the FR regions and, together with the CDRs of the other chain, contribute to the formation of the antibody's antigen-binding site.

[0076] In some embodiments, the heavy chain antibody of this disclosure or its VHH backbone region includes FR1, FR2, FR3, and FR4 regions, the sequences of which are shown in the FR region sequences of VHH as shown in any of SEQ ID NO:49-96. The sequences of the aforementioned FRs can be arbitrarily combined for use in the CD19 heavy chain antibody, CD22 heavy chain antibody, or BCMA heavy chain antibody of this disclosure. Exemplarily, the backbone region of the CD19 heavy chain antibody or VHH of this disclosure includes the FR region of VHH as shown in any of SEQ ID NO:49-59. The backbone region of the CD22 heavy chain antibody or VHH of this disclosure includes the FR region of VHH as shown in any of SEQ ID NO:60-74. The backbone region of the anti-BCMA heavy chain antibody of this disclosure includes the FR region of VHH as shown in any of SEQ ID NO:75-82. The FR region of the VHH includes a wild-type FR region and a humanized FR region.

[0077] The “Fc region” (crystallizable fragment region), “Fc domain”, or simply “Fc” refers to the C-terminal region of an antibody heavy chain, which mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. Fc receptors are immunoglobulin family proteins expressed on the surface of specific immune cells that recognize antibody Fc regions to mediate immune responses. After the antibody Fab region recognizes an antigen, its antibody Fc region binds to Fc receptors on immune cells (e.g., killer cells), initiating immune cell responses such as phagocytosis and ADCC. The human IgG family comprises four members: IgG1, IgG2, IgG3, and IgG4. The amino acid differences in the crystallizable (Fc) region of their heavy chain constant regions result in varying affinities for FcγRs. To enhance cell-killing efficacy, multispecific antibodies or their functional regions may possess native Fc sequences or variant Fc sequences.

[0078] An "antibody fragment" comprises a portion of a complete antibody, preferably the antigen-binding region and / or variable region of the complete antibody. The antibody fragment is preferably an antigen-binding fragment of the antibody. Examples of heavy chain antibody fragments include Fv fragments; biantibodies; linear antibodies; single-domain antibodies (VHH); single-chain antibody molecules; scFv-Fc fragments; and any fragment that should be able to increase its half-life through chemical modification or incorporation into liposomes.

[0079] "Fv" is the smallest antibody fragment containing complete antigen recognition and binding sites. For heavy chain antibodies, this fragment consists of heavy chain variable domains. "Single-chain Fv" can also be abbreviated as "sFv" or "scFv", and is an antibody fragment containing the VHH domain of the heavy chain antibody linked together to form a single polypeptide chain.

[0080] In this document, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that the individual antibodies constituting the group are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site. Compared to polyclonal antibody formulations (which typically consist of different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage of being synthesized through hybridoma culture, free from contamination by other immunoglobulins. The modifier "monoclonal" indicates the characteristic that the antibody is obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used according to this disclosure can be generated by a variety of techniques, including, for example, hybridoma methods, phage display methods, recombinant DNA methods, and techniques for generating human or human-like antibodies from animals having partial or whole human immunoglobulin loci or genes encoding human immunoglobulin sequences, single-cell sequencing methods.

[0081] Monoclonal antibodies also include “chimeric” antibodies in this article, wherein a portion of the chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided they exhibit the desired biological activity.

[0082] The “humanized” form of a non-human (e.g., mouse) antibody refers to a chimeric antibody that contains at least a sequence derived from a non-human immunoglobulin. Therefore, a “humanized antibody” generally refers to a non-human antibody whose variable domain backbone region is exchanged with a sequence found in human antibodies. Typically, in a humanized antibody, the entire antibody (except for the CDR) is encoded by a human-derived polynucleotide or is identical to that antibody (except for the CDR). The CDR (some or all of which are encoded by nucleic acids derived from non-human organisms) is transplanted into the β-sheet backbone of the variable region of the human antibody to produce an antibody whose specificity is determined by the transplanted CDR. Methods for producing such antibodies are well known in the art, for example, using mice with genetically engineered immune systems.

[0083] "Human antibody" refers to an antibody having an amino acid sequence corresponding to that of antibodies generated by humans and / or produced using any of the techniques disclosed herein for generating human antibodies. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries.

[0084] In some embodiments, the antigen-binding fragment of the anti-CD19 heavy chain antibody is the CD19 binding molecule of any embodiment of CN202410429873.8, the entire contents of which are incorporated herein by reference. Preferably, the antigen-binding fragment of the anti-CD19 heavy chain antibody has the sequence shown in any one of SEQ ID NO:49-59 or a sequence having at least 80% sequence identity with it, wherein each CDR sequence is identical to any one of SEQ ID NO:49-59.

[0085] In some embodiments, the antigen-binding fragment of the anti-CD22 heavy chain antibody is the CD22-binding molecule of any embodiment of CN202410429871.9, the entire contents of which are incorporated herein by reference. Preferably, the antigen-binding fragment of the anti-CD22 heavy chain antibody has a sequence shown in any of SEQ ID NO:60-74 or a sequence having at least 80% sequence identity with it, wherein each CDR sequence is identical to any of SEQ ID NO:60-74.

[0086] In some embodiments, the antigen-binding fragment of the anti-BCMA heavy chain antibody is the BCMA-binding molecule of any embodiment of PCT / CN2022 / 082351, the entire contents of which are incorporated herein by reference. Preferably, the antigen-binding fragment of the anti-BCMA heavy chain antibody has a sequence shown in any of SEQ ID NO:75-82 or a sequence having at least 80% sequence identity with it, wherein each CDR sequence is identical to any of SEQ ID NO:75-82.

[0087] The first functional region targeting CD19 described herein may be a monovalent or multivalent single-domain antibody, multispecific single-domain antibody, heavy chain antibody, or antigen-binding fragment thereof comprising one, two, or more anti-CD19 single-domain antibodies described herein. The second functional region targeting CD22 described herein may be a monovalent or multivalent single-domain antibody, multispecific single-domain antibody, heavy chain antibody, or antigen-binding fragment thereof comprising one, two, or more anti-CD22 single-domain antibodies described herein. The third functional region targeting BCMA described herein may be a monovalent or multivalent single-domain antibody, multispecific single-domain antibody, heavy chain antibody, or antigen-binding fragment thereof comprising one, two, or more anti-BCMA single-domain antibodies described herein. Anti-CD19 heavy chain antibodies, anti-CD22 heavy chain antibodies, and / or anti-BCMA heavy chain antibodies also include a heavy chain constant region, such as the constant region of a camel heavy chain antibody or a cartilaginous fish heavy chain antibody.

[0088] This disclosure also includes the antibody derivatives and analogs described herein. “Derivatives” and “analytes” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of this disclosure. The derivatives or analogs of this disclosure may be (i) polypeptides having substituents in one or more amino acid residues, or (ii) polypeptides formed by fusing a mature polypeptide with another compound (such as a compound that extends the half-life of the polypeptide, e.g., polyethylene glycol), or (iii) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these derivatives and analogs are within the scope well known to those skilled in the art.

[0089] Without substantially affecting antibody activity, those skilled in the art can modify the sequence of this disclosure by one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) to obtain variants of the antibody or its functional fragment sequence. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus. In the art, conservative substitutions with amino acids of similar or comparable properties generally do not alter protein function. For example, substitutions of amino acids with similar properties in the FR and / or CDR regions of the variable region. Amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. For example, adding one or more amino acids to the C-terminus and / or N-terminus generally does not change the function of the protein. These are all considered to be included within the scope of protection of this disclosure.

[0090] The variants of antibodies described herein include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibodies of this disclosure under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the antibodies of this disclosure.

[0091] In some embodiments, the sequence of the variants described herein may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with its source sequence. This sequence identity can be measured using sequence analysis software, such as the computer program BLAST with default parameters, particularly BLASTP or TBLASTN. This disclosure also includes molecules having a variable region of the antibody heavy chain with a CDR, provided that its CDR has at least 90% (preferably at least 95%, most preferably at least 98%) homology with the CDR identified herein.

[0092] In some embodiments, the multispecific antibody contains any of the sequences shown in SEQ ID NO:83-96.

[0093] In some embodiments, the multispecific antibody also contains an Fc region; preferably, the Fc region is the Fc region of IgG1, IgG2, IgG3 or IgG4; wherein the IgG1, IgG2, IgG3 or IgG4 is human-derived.

[0094] Because the Fc region can cause significant ADCC and CDC effects, potentially leading to immune cell damage and exhibiting negative pharmacological effects, Fc is often modified (referred to as variant Fc in this article). Through site mutations, the affinity constant of multispecific antibodies for FcγRIIIa and / or C1q is reduced compared to the original value, thereby improving the efficacy of antibody drugs. Currently disclosed mutation sites, according to the EU numbering system, include: IgG1 Fc region mutation sites include L234A, L235A, L235E, L235G, G236A, G237A, N297A, G318A, L320A, and L322A; IgG3 Fc region mutation sites include V234A, G237A, P238S, H28A, and V309L. The mutation sites in the Fc region of IgG3 include Leu281, Leu282, Gly283, Gly284, Asn344, and Pro378; the mutation sites in the Fc region of IgG4 include S228P, E233P, F234V, L235A, F243L, D254A, R292P, Y300L, L309V, and R409K. The variant Fc in this article includes, but is not limited to, the Fc of each IgG having the above-mentioned mutation sites.

[0095] In some embodiments, the Fc region is the Fc region of IgG1, and according to the EU numbering system, the Fc region has one or more of the following mutations: L234A, L235A, G237A. In some embodiments, the Fc region is the Fc region of IgG4, and according to the EU numbering system, the Fc region has one or more of the following mutations: S228P, E233P, F234V, L235A, D254A, L309V, R409K.

[0096] The antibodies of this disclosure can be prepared using methods conventional in the art, such as hybridoma techniques well known in the art. The heavy chain antibodies of this disclosure can be prepared using methods conventional in the art, such as phage display techniques well known in the art. Alternatively, the antibodies or heavy chain antibodies of this disclosure can be expressed in other cell lines. Suitable mammalian host cells can be transformed with sequences encoding the antibodies of this disclosure. Transformation can be performed using any known method, including, for example, packaging polynucleotides in a virus (or viral vector) and transducing host cells with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Mammalian cell lines suitable as hosts for expression are well known in the art, including but not limited to a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS) cells, and human hepatocellular carcinoma cells (e.g., HepG2). Particularly preferred cell lines are selected by identifying which cell lines exhibit high expression levels and produce antibodies with basic CD19, CD22, and BCMA binding properties.

[0097] As those skilled in the art will recognize, immunoconjugates and fusion expression products include conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to antibodies or fragments thereof as disclosed herein. This disclosure also includes cell surface markers or antigens that bind to said antibodies or fragments thereof.

[0098] This disclosure also includes fusion proteins containing the multispecific antigen-binding molecule described herein and other peptides. In some embodiments, the other peptides are located at the N-terminus and / or C-terminus of the binding molecule. In some embodiments, the other peptides include peptides that localize the binding molecule to different organelles, tags for purification or for immune responses, transmembrane proteins or their transmembrane regions, chimeric antigen receptors or components thereof (extracellular domains, hinge regions, transmembrane regions, signal transduction domains, co-stimulatory domains, etc.). In some embodiments, the fusion protein is a chimeric antigen receptor whose antigen-binding domain contains the multispecific antigen-binding molecule. The chimeric antigen receptor comprises: an optional signal peptide sequence, a multispecific antigen-binding molecule, a hinge region, a transmembrane region, and an intracellular region.

[0099] The chimeric antigen receptor also has one or more of the following characteristics:

[0100] The signal peptide includes CD8 signal peptide, CD28 signal peptide, CD4 signal peptide, or light chain signal peptide.

[0101] The hinge region includes a CD8 hinge region, an IgD hinge region, an IgG1 Fc CH2CH3 hinge region, or an IgG4 Fc CH2CH3 hinge region.

[0102] The transmembrane regions include CD28 transmembrane regions, CD8 transmembrane regions, CD3ζ transmembrane regions, CD134 transmembrane regions, CD137 transmembrane regions, ICOS transmembrane regions, or DAP10 transmembrane regions.

[0103] The intracellular region includes an intracellular co-stimulatory domain and / or an intracellular signaling domain.

[0104] The intracellular co-stimulatory domains include the intracellular domains of CD28, CD134 / OX40, CD137 / 4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T cell co-stimulatory factor (ICOS), or DNAX activator protein 10.

[0105] The intracellular signaling domain includes the CD3ζ intracellular signaling domain or the FcεRIγ intracellular signaling domain.

[0106] Nucleic acid

[0107] This disclosure also provides polynucleotides encoding multispecific antigen-binding molecules or fusion proteins thereof. This document provides polynucleotides encoding the heavy chain variable region, the heavy chain, and each CDR. The polynucleotides of this disclosure may be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or synthetically produced DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0108] As those skilled in the art will understand, due to the degeneracy of the genetic code, extremely large quantities of nucleic acids can be produced, all of which encode the antibodies or antigen-binding fragments of this disclosure. Therefore, given the identification of specific amino acid sequences, those skilled in the art can produce any number of different nucleic acids by simply modifying the sequence of one or more codons without altering the amino acid sequence encoding the protein. Thus, this disclosure also relates to polynucleotides that hybridize with the aforementioned polynucleotide sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. This disclosure particularly relates to polynucleotides that hybridize with the polynucleotides described in this disclosure under stringent conditions. In this disclosure, "strict conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the similarity between the two sequences is at least 90%, preferably more than 95%. Furthermore, the hybridizable polynucleotide-encoded polypeptide has the same biological function and activity as the mature polypeptide.

[0109] The full-length nucleotide sequence or fragments of the antibodies disclosed herein can typically be obtained using PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequences artificially, especially when the fragment length is short. Generally, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0110] Once the relevant sequence is obtained, it can be mass-produced using recombinant methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this disclosure include biomolecules existing in isolated forms. Currently, DNA sequences encoding the proteins (or fragments thereof, or derivatives thereof) of this disclosure can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. Furthermore, mutations can be introduced into the protein sequences of this disclosure through chemical synthesis.

[0111] Therefore, this disclosure also relates to nucleic acid constructs, such as expression vectors and recombinant vectors, that contain the appropriate DNA sequences described above and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins. Vectors typically contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. These sequences (collectively referred to as “flanking sequences” in some embodiments) typically include one or more of the following nucleotide sequences: promoter, one or more enhancer sequences, origin of replication, transcription termination sequence, complete intron sequences containing donor and acceptor splicing sites, sequence encoding a leader sequence for polypeptide secretion, ribosome binding site, polyadenylated sequence, multi-linker regions for inserting nucleic acids encoding antibodies to be expressed, and optional marker elements.

[0112] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0113] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0114] The fusion protein is a chimeric antigen receptor (CAR), and its antigen-binding domain contains the bispecific binding molecule. Cells expressing the chimeric antigen receptor can be activated by: (1) contacting the cells with an activator; (2) contacting the cells with a nucleic acid molecule encoding the CAR, the nucleic acid molecule encoding the CAR being on a vector, to introduce the nucleic acid molecule into the cells; and (3) harvesting the cells. The method can employ conventional preparation processes, for example, a total preparation time of 9 days or longer; the method can also employ rapid processes, for example, preparation times of 30 h, 24 h, 6 h or less. In some embodiments, it can be prepared according to the method of any embodiment of PCT / CN2024 / 130978, the entire contents of which are incorporated herein by reference.

[0115] Cells expressing chimeric antigen receptors can be prepared in vitro using viral or non-viral vectors; or cells expressing chimeric antigen receptors can be directly generated in the patient's body using viral or non-viral vectors. In some embodiments, the nucleic acid molecule encoding CAR is DNA, and the vector is a plasmid vector. In some embodiments, the plasmid vector is an antibiotic-free microplasmid; for antibiotic-free microplasmids applicable to this invention, please refer to patent applications PCT / CN2024 / 072052 and CN202410952401.0, the entire contents of which are incorporated herein by reference. In some embodiments, the nucleic acid molecule encoding CAR is RNA, such as mRNA or saRNA, and the vector is LNP, LPX, VLP, inorganic nanoparticles, or exosomes. In some embodiments, the vector is a plasmid vector containing a transposon containing a nucleic acid molecule encoding CAR, and the cells in step (2) are also in contact with a transposase or a nucleic acid molecule encoding a transposase. The transposon and transposase belong to the same transposon system, which is selected from: the Tol1 transposon system, the Tol2 transposon system, the Frog Prince transposon system, the Minos transposon system, the Hsmar1 transposon system, the Helraiser transposon system, the ZB transposon system, the BZ transposon system, the Intruder transposon system, the SPINON transposon system, the TcBuster transposon system, the Passer transposon system, the JL transposon system, the Yabusame-1 transposon system, the Uribo2 transposon system, the PiggyBac (PB) transposon system, the SleepingBeauty (SB) transposon system, and various variants or derivatives of the above transposon systems. In one or more embodiments, the transposon system is the PB transposon system, the BZ transposon system (PCT / CN2023 / 120383), or the JL transposon system (PCT / CN2024 / 076381). In some embodiments, the nucleic acid molecule encoding the transposase is DNA or RNA. In some embodiments, cells are contacted with the transposase or the nucleic acid molecule encoding the transposase, and cell transduction is performed by electroporation. In some embodiments, the introduction is performed by electroporation. Step (2) includes contacting cells with a DNA vector containing a PB transposon and mRNA encoding the PB transposase, the PB transposon comprising a CAR gene expression cassette and terminal inverted repeat sequences flanking the CAR gene expression cassette. Methods for in vivo preparation of CAR-T cells can be employed using methods known in the art. In some embodiments, the CAR gene expression cassette is loaded onto a viral vector infecting T cells, and the viral vector is injected into a patient to directly generate CAR-T cells in vivo. In some embodiments, CAR-T cells are directly generated in vivo by delivering CAR mRNA using LNP.Antibody fragments can also be fused to LNPs to generate more precise targeting LNPs, which can be used to deliver CAR mRNA into the body for the instantaneous preparation of CAR-T.

[0116] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this disclosure. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0117] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0118] This document also provides a method for producing the multispecific antigen-binding molecule or its fusion protein as claimed, comprising: culturing the host cells described herein under conditions suitable for producing the multispecific antigen-binding molecule or the fusion protein, and optionally purifying the multispecific antigen-binding molecule from the culture, or incubating a nucleic acid molecule encoding the multispecific antigen-binding molecule or the fusion protein under conditions suitable for translating DNA or RNA in a non-cellular system (e.g., solution).

[0119] Therapeutic Uses and Pharmaceutical Compositions

[0120] All aspects of the antibodies described herein can be used to prepare drugs for the prevention or treatment of the various conditions and diseases described herein, particularly those related to cells expressing CD19 and / or CD22. In some implementations, the condition and disease are cancers, including but not limited to: B-cell acute lymphoblastic leukemia (“BALL”), T-cell acute lymphoblastic leukemia (“TALL”), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), multiple myeloma, follicular lymphoma, spleen, marginal zone lymphoma, mantle cell lymphoma, indolent B-cell lymphoma or Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, relapsed or refractory acute lymphoblastic leukemia (r / r ALL), relapsed or refractory diffuse large B-cell lymphoma (r / r DLBCL), relapsed or refractory follicular lymphoma (r / r FL), etc.

[0121] The pharmaceutical compositions described herein contain the binding molecules described herein, as well as pharmaceutically acceptable excipients, including but not limited to diluents, carriers, solubilizers, emulsifiers, preservatives, and / or adjuvants. The excipients are preferably non-toxic to the recipient at the doses and concentrations used. Such excipients include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. In some embodiments, the pharmaceutical composition may contain substances for improving, maintaining, or retaining, for example, the composition's pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or permeation. These substances are known in the art. The optimal pharmaceutical composition can be determined based on the intended route of administration, delivery method, and required dosage.

[0122] Pharmaceutical compositions intended for internal administration are typically provided in sterile formulations. Sterilization is achieved by filtration through a sterile filter membrane. Sterilization can be performed using this method before or after lyophilization and rehydration when the composition is lyophilized. The pharmaceutical compositions of this disclosure may be used for parenteral delivery. Compositions intended for parenteral administration may be lyophilized or stored in solution. They may be prepared, for example, using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. Parenteral compositions are typically placed in containers with sterile access openings, such as intravenous solution bands or vials with stoppers that can be punctured by a hypodermic needle. Alternatively, the compositions may be intended for inhalation or delivery via the digestive tract (e.g., orally). The preparation of the pharmaceutically acceptable compositions is within the scope of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations containing antibodies in sustained or controlled-release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery methods, such as liposome carriers, bioeasily perishable microparticles or porous beads, and accumulation injection, are also known to those skilled in the art.

[0123] Once formulated, the pharmaceutical composition is stored in sterile vials as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. The formulation may be stored in a ready-to-use form or rehydrated before administration (e.g., lyophilized). This disclosure also provides kits for generating single-dose administration units. The kits of this disclosure may each contain a first container with dried protein and a second container with an aqueous formulation. In some embodiments of this disclosure, kits containing single-lumen and multi-lumen pre-filled syringes (e.g., liquid syringes and lyophilized syringes) are provided.

[0124] This disclosure also provides methods for treating patients (particularly patients with CD19, CD22, and / or BCMA-related diseases) by administering the binding molecule or pharmaceutical composition thereof described in any embodiment of this disclosure. In this document, the terms “patient,” “subject,” “individual,” and “object” are used interchangeably and include any living organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, etc.), and most preferably a human. “Treatment” refers to a subject receiving a treatment regimen described herein to achieve at least one positive therapeutic effect (e.g., a reduction in the number of cancer cells, a reduction in tumor volume, a decrease in the rate of cancer cell invasion into surrounding organs, or a decrease in the rate of tumor metastasis or tumor growth). Effective treatment regimens for patients can vary depending on various factors (e.g., the patient’s disease status, age, weight, and the ability of the therapy to elicit an anticancer response in the subject). In some embodiments, the method includes administering a therapeutically effective amount of CAR-T cells to a patient in need, the CAR-T cells expressing an antigen-binding domain of a chimeric antigen receptor containing the multispecific antigen-binding molecule, preferably, the amino acid sequence of the chimeric antigen receptor as shown in SEQ ID NO: 89. In some implementations, lymphocyte depletion may or may not be performed on the patient prior to CAR-T cell administration, preferably without lymphocyte depletion. The CAR-T cells provided in this disclosure can be administered without lymphocyte depletion, with minimal impact on treatment efficacy, and can reduce toxicity and infection risks.

[0125] The therapeutically effective amount of the pharmaceutical composition containing the molecules described herein will depend, for example, on the extent of treatment and the target. Those skilled in the art will understand that the appropriate dose level for treatment will vary in part depending on the delivered molecule, indication, route of administration, and the patient's size (weight, body surface or organ size) and / or condition (age and general health status). In some embodiments, clinicians may titrate the dose and change the route of administration to obtain optimal therapeutic effect. For example, approximately 10 micrograms / kg body weight to approximately 50 mg / kg body weight per day.

[0126] The frequency of administration will depend on the pharmacokinetic parameters of the bound molecules in the formulation used. Clinicians typically administer the composition until a dose is reached to achieve the desired effect. The composition can therefore be administered as a single dose, or over time as two or more doses (with or without the same amount of the desired molecule), or via implanted device or catheter as a continuous infusion.

[0127] The drug composition is administered via known methods, such as oral, intravenous, intraperitoneal, intracerebral (within brain parenchyma), intraventricular, intramuscular, intraocular, intraarterial, portal vein, or intralesional injection; via a continuous release system or via an implanted device.

[0128] Diagnostics, tests and kits

[0129] The binding molecules disclosed herein, due to their high affinity for CD19, CD22, and BCMA, can be used for assays, such as binding assays, to detect and / or quantify CD19, CD22, and BCMA expressed in tissues or cells. Binding molecules, such as single-domain antibodies, can be used in studies further investigating the role of CD19, CD22, and BCMA in disease. The methods for detecting CD19, CD22, and BCMA are generally as follows: obtaining cell and / or tissue samples; detecting the levels of CD19, CD22, and BCMA in the samples.

[0130] The multispecific antibodies disclosed herein can be used for diagnostic purposes to detect, diagnose, or monitor diseases and / or conditions associated with CD19, CD22, and BCMA. This disclosure provides methods for detecting the presence of CD19, CD22, and BCMA in samples using classical immunohistochemical methods known to those skilled in the art. Detection of CD19, CD22, and BCMA can be performed in vivo or in vitro. Examples of methods suitable for detecting the presence of CD19, CD22, and BCMA include ELISA, FACS, and RIA.

[0131] For diagnostic applications, binding molecules are typically labeled with detectable labeling groups, such as single-domain antibodies. Suitable labeling groups include (but are not limited to) the following: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinylated groups, or predetermined polypeptide epitopes recognized by secondary reporter molecules (e.g., leucine zipper pairs, binding sites for secondary antibodies, metal-binding domains, epitope tags), MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents. Various methods for labeling proteins are known in the art and can be used for the purposes of this disclosure.

[0132] Another aspect of this disclosure provides a method for detecting the presence of a test molecule that competitively binds to CD19, CD22, and BCMA with an antibody of this disclosure. An example of such an assay would involve detecting the amount of free antibody in a solution containing a certain amount of CD19, CD22, and BCMA, in the presence or absence of the test molecule. An increase in the amount of free antibody (i.e., antibody not bound to CD19, CD22, and BCMA) would indicate that the test molecule is able to competitively bind to CD19, CD22, and BCMA with the antibody. In one embodiment, the antibody is labeled with a labeling group. Alternatively, the test molecule is labeled and the amount of free test molecule is monitored in the presence or absence of the antibody.

[0133] This disclosure also provides a detection kit for detecting CD19, CD22, and BCMA levels. The kit includes the multispecific antibody described herein, a lysis medium for dissolving samples, and universal reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. This detection kit can be used as an in vitro diagnostic device.

[0134] The embodiments of this disclosure will be described in detail below. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of this disclosure. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased on the market.

[0135] Example

[0136] Among the nanobodies provided in this embodiment, NBL508-170, NBL508-10, NBL508-24, NBL508-20, and NBL508-53 are anti-CD19 VHH; NBL526-590, NBL526-998, NBL526-C44, NBL526-S3-C84, NBL526-C5, NBL526-1011, and NBL526-S3-648 are anti-CD22 VHH; and NB-257, NB-192, NB-34, and NB-27 are anti-BCMA VHH. Their sequences are shown in Table 2.

[0137] Table 2: Anti-CD19, anti-CD22 and anti-BCMA VHH sequences

[0138] The CDR regions of the above-mentioned anti-CD19, anti-CD22 and anti-BCMA VHH are shown in Table 3.

[0139] Table 3: CDR regions of anti-CD19, anti-CD22 and anti-BCMA VHH

[0140] After humanization, the nanobodies underwent mutation in the FR region while the CDR region remained unchanged. The amino acid sequences of the anti-CD19 VHH were any of SEQ ID NO:49-59, the anti-CD22 VHH were any of SEQ ID NO:60-74, and the anti-BCMA VHH were any of SEQ ID NO:75-82. The original or humanized anti-CD19 VHH, anti-CD22 VHH, and anti-BCMA VHH were then used as linkers to construct trispecific antibodies. The amino acid sequences of the trispecific antibodies used in this embodiment are shown in Table 4.

[0141] Table 4: Names and sequences of trispecific antibodies

[0142] The present disclosure will be described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the present disclosure. Unless otherwise stated, the methods and materials used in the embodiments are conventional materials and methods in the art.

[0143] Example

[0144] Example 1: Expression and purification of candidate antibodies

[0145] The trispecific antibodies in Table 4 were prepared by Baiying Biotechnology, expressed on HEK293, purified using a Ni column, and stored in PBS. Protein concentration was then detected using Nanodrop, and protein purity was determined by HPLC. The obtained protein purity and yield met the requirements for subsequent experiments.

[0146] Example 2, Characterization of Triple Antibody Candidates

[0147] (1) Protein-level affinity assay: Surface plasmon resonance (SPR) was used to determine the binding kinetics and affinity of heavy chain antibodies for human CD19, CD22, and BCMA antigens. Purified antibodies were flowed through a sensor chip pre-immobilized with protein A, where they were captured. Two different concentrations of CD19, CD22, and BCMA proteins were used as the mobile phase, with binding and dissociation times of 120 s and 1200 s, respectively. The binding rate (kon), dissociation rate (koff), and equilibrium constant (KD) were analyzed using Biacore Evaluation Software 2.0 (GE), and the results are shown in Table 5.

[0148] Table 5: Affinity Detection of Triple Antibody Protein Levels

[0149] (2) Cell-level affinity assay: Raji WT, Raji CD19 KO, Raji CD22 KO, and RPMI8226 cells were seeded in 96-well plates, with 3 × 10⁶ cells per well. 5 Cells were then incubated with serially diluted trispecific antibodies for half an hour. After incubation, the secondary antibody anti-human IgG PE (Jackson Immuno Research, Code: 109-117-008, Lot: 145501) was added and incubated again. Detection was then performed using a CytoFLEX flow cytometer. The EC50 of the antibodies was calculated using a fitted curve. The two batches of antibodies were identified separately, and the results are shown in Figures 1-7 and Table 6. In Table 6, " / " indicates no detection.

[0150] Table 6: EC50 of triple antibody binding to cells

[0151] Example 3, Tissue cross-reactivity of trispecific antibodies

[0152] Thirty-four tissue samples were selected for frozen sectioning, air-dried at room temperature, and fixed with acetone. Blocking was performed using reagents A and B of the endogenous biotin blocking kit (Sangon Biotech, E674001). Biotin-labeled antibody samples were incubated for 30 min, washed, and then incubated for 15 min with horseradish peroxidase-labeled streptavidin (Abcam, ab7403). DAB staining and hematoxylin counterstaining were performed, followed by mounting on neutral plastic and air-drying for microscopic examination. The CD19 positive control was CD19 antibody (Novus) (NBP1-43436), the CD22 positive control was Abcam Anti-CD22 antibody (ab112182), the BCMA positive control was Abcam Anti-BCMA antibody (ab245940), and the negative control was Abcam antibody (ab200208). The results are shown in Table 7 (NO. in Table 7 refers to the tissue source number), indicating that most antibodies showed good specificity in normal tissues, except for some positive staining in some lymphoid follicle tissues. All the antibodies tested showed negative reactions in the parathyroid glands after re-examination.

[0153] Table 7: Tissue Cross-Reactivity of Trispecific Antibodies

[0154] Example 4: Preparation and performance evaluation of trispecific CAR-T

[0155] (1) Preparation of CAR-T cells

[0156] CAR-T cells were prepared using trispecific antibodies 9B2-P1-1-E1 and 29B-P1-1-F5 as the antigen-binding domains of chimeric antigen receptors. The CAR structure consisted of, from the N-terminus to the C-terminus, a CD8α signal peptide (SEQ ID NO: 98), a trispecific antibody (SEQ ID NO: 89 or 90), a CD8α hinge region (SEQ ID NO: 99), a CD8 transmembrane region (SEQ ID NO: 100), a 4-1BB intracellular co-stimulatory signaling region (SEQ ID NO: 101), and a CD3ζ intracellular signaling domain (SEQ ID NO: 102), as shown in Figure 8. The prepared CAR-T cells were abbreviated as "E1" and "F5," respectively. CAR-T cells targeting CD19, CD22, and BCMA monospecific antibodies as antigen-binding domains and CAR-T cells without CARs were used as controls and named CD19 BMK, CD22 BMK, BCMA BMK, and Mock-T, respectively. Among them, the CD19 BMK monospecific antibody is FMC63, the CD22 BMK monospecific antibody is m971 antibody, and the BCMA BMK monospecific antibody is BCMA antibody (CN106795217B) from Bluebird Biotechnology Co., Ltd.

[0157] The preparation includes the following steps:

[0158] ① A single white blood cell sample was obtained from the patient, and then T cells were sorted using Miltenyi CD4 / CD8 magnetic beads, 1×10⁻⁶. 9 After adding 200 μL of CD4 magnetic beads and 200 μL of CD8 magnetic beads to WBC and incubating for 30 min, CD4 was screened using an XS sorting column. + T and CD8 + T cells.

[0159] ② After sorting, take 1.05×10 8 Cell suspension was transferred to coated bottles containing Miltenyi MACS GMP TransAct CD3 / 28 magnetic beads, and culture medium (AIM-V + 5% SR) was added to 30 mL / bottle. IL-7 and IL-15 were added to a final concentration of 25 ng / mL and 25 ng / mL, respectively. The bottles were then incubated at 37°C with 5% CO2 for 1–48 h.

[0160] ③ Collect T cells 24 hours after activation, 1*10 7Cells / groups were treated with 5 μg / mL of plasmid expressing a CAR containing trispecific antibodies and 100 μg / mL of JL transposase mRNA. The mixture was transferred to an electroporation cuvette and placed in a Lonza Nucleofactor 4D or Maxcyte electroporator. Electroporation was performed using program FI-115 or Resting T / Expand T4. The electroporated cell suspension was then transferred to a 37°C incubator and cultured for 30 min. After culture, the cells were either cryopreserved or subjected to quality control testing.

[0161] ④ Prepare the cryopreservation solution with the following formula: compound electrolyte injection: dextran 40 glucose injection: human serum albumin: DMSO = 65:10:20:5. Cryopreserve the CAR-T cells cultured for 30 min.

[0162] The backbone sequence of the plasmid expressing CAR is shown in SEQ ID NO: 103; the amino acid sequence of the JL transposase is shown in SEQ ID NO: 104.

[0163] (2) CAR-T cell positivity rate

[0164] After the prepared CAR-T or cryopreserved CAR-T was revived, it was cultured in AIMV CTS medium containing 50 ng / mL IL7, 50 ng / mL IL15 and 10% FBS. On day 4, approximately 1E5 total viable cells of each of the five CAR-T cell types were collected, centrifuged at 1500g for 1 min, and the cell supernatant was discarded. The cell pellet was resuspended in 200 μL of 1×staining buffer and centrifuged at 1500g for 1 min. The cell pellet was then resuspended in 200 μL of 1×staining buffer and centrifuged at 1500g for 1 min. The cell pellet was then resuspended in 100 μL of 1×staining buffer. 1 μL of anti-CD3 and anti-VHH (GenScript, catalog number A02227) antibodies were added, mixed well, and incubated in the dark for 30 min. After incubation, the cells were washed three times with 200 μL of 1×staining buffer, and finally resuspended in 100 μL of 1×staining buffer for flow cytometry analysis. The positive rates of the prepared CAR-T cells are shown in Figure 9. Among them, the positive rate of E1 is the highest (78.3%), the positive rate of F5 is comparable to that of CD19 BMK, and the positive rates of CD22 BMK and BCMA BMK are slightly lower, but all are higher than 45%.

[0165] (3) Phenotype of CAR-T cells

[0166] Following the method in Example 3 of CN202311496656.2, cell differentiation phenotypes were analyzed using flow cytometry. The memory phenotype clustering results are shown in Figure 10, where TSCM:CD95 + CD45RO - CCR7 + TCM:CD45RO + CCR7 + TEM:CD45RO + CCR7 - Teff:CD45RO - CCR7 - The results showed that the memory phenotypes of different CAR-T cells were not significantly different, with the Tcm ratio of trispecific CAR-T cells being slightly higher than that of monoclonal antibody CAR-T cells. The proportions of CD4 and CD8 cells were similar among different CAR-T cells. This indicates that the preparation of trispecific CAR-T cells does not lead to significant changes in their cell typing.

[0167] (4) Multi-round co-culture of tumor cells

[0168] 5000 target cells of each of the four types were resuspended in 50 μL of culture medium and cultured for approximately 24 hours. Once the cell index (CI, indicating cell growth) reached approximately 1.5, different numbers of effector cells (CAR-T cells) were resuspended in 50 μL of culture medium at an effector-to-target ratio of 1:4 and co-cultured with tumor cells for 4 days. Five rounds of culture were performed, each lasting 4 days.

[0169] Using co-culture of tumor cells, after repeated stimulation of CAR-T cells, three types of tumor cells (CD22) were observed. + Raji cells, CD19 + The results for Raji cells and RPMI-8226 cells are shown in Figures 11-13. The AD values ​​in the figures represent, in order, the tumor cell killing rate, changes in CAR positivity rate, CAR-T cell proliferation fold increase, and IFN-γ secretion results.

[0170] Based on the results, compared to CD22 + Raji cell co-culture (Figure 11) showed that, compared with CD22BMK, trispecific CAR-T cells had lower tumor cell killing and IFN-γ levels with increasing co-culture cycles, but had significant advantages in CAR positivity rate and CAR-T cell proliferation.

[0171] With CD19 + Raji cells co-culture (Figure 12) showed similar effects to CD19 BMK in trispecific CAR-T therapy.

[0172] When co-cultured with RPMI-8226 cells (Figure 13), the trispecific CAR-T showed significant advantages over BCMA BMK in all aspects as the number of co-culture cycles increased, especially in the 3rd and 4th cycles.

[0173] (5) Preclinical research

[0174] WT Raji tumor cells (0.5*10) 6 The sample was resuspended in 100 μL of PBS, mixed thoroughly with 100 μL of matrix gel (BD), and subcutaneously injected into the back of NPSG-severely immunodeficient mice (nine mice per group). Tumors were allowed to develop after reaching a tumor volume of 100-150 mm. 3 Vehicle, MOCK-T, E1 CAR-T, and F5 CAR-T were injected at different times, and the number of CAR-T positive cells was 1×10⁻⁶. 7 Each sample was resuspended in 200 μL of PBS and administered intravenously to mice in a single dose. The body weight of the mice 49 days after the single injection is shown in Figure 14. No mice died or showed significant weight loss, indicating high in vivo safety.

[0175] Vehicle, MOCK-T, and E1 CAR-T are designed with three different injection doses: high, medium, and low, each 3.3 × 10⁻⁶. 6 1×10 6 The sum is 0.1 × 10 6 Each tumor sample was resuspended in 200 μL of PBS and injected into mice via a single intravenous infusion. Mice were observed weekly for four weeks, and tumor growth was assessed using fluorescence detection. The results are shown in Figure 15. From day 7 onwards, tumors at high concentrations (3.3 × 10⁻⁶) showed significant growth. 6 ) and medium (1×10 6 At high doses, E1 CAR-T showed good efficacy, and even at low doses, it showed some efficacy. Therefore, trispecific CAR-T significantly inhibited the growth of CD19+CD22+ tumors in a dose-dependent manner, with a minimum effective dose of 0.1×10⁻⁶. 6 CAR-T cells / mouse. At high doses, compared with CD19 CAR-T (BMK), as shown in Figure 16, E1 CAR-T showed superior tumor-suppressing effects.

[0176] The number of positive CAR-T cells in mouse peripheral blood was detected to assess the CAR-T amplification capacity, and the results are shown in Figure 17. The amplification capacity of E1 CAR-T (BZE2204) was significantly better than that of CD19 CAR-T (BMK).

[0177] Example 4: Preparation and performance evaluation of CAR-T cells prepared using a 6-hour process.

[0178] (1) The steps of the 6h preparation process are as follows:

[0179] ① A single white blood cell sample was obtained from the patient, and then T cells were sorted using Miltenyi CD4 / CD8 magnetic beads, 1×10⁻⁶. 9 After adding 200 μL of CD4 magnetic beads and 200 μL of CD8 magnetic beads to WBC and incubating for 30 min, CD4 was screened using an XS sorting column. + T and CD8 + T cells.

[0180] ② After sorting, take 1.05×10 8 Cell suspension was transferred to a coated bottle containing Miltenyi MACS GMP TransAct CD3 / 28 magnetic beads, and culture medium (AIM-V + 5% SR) was added to 30 mL / bottle. IL-7 and IL-15 were added to a final concentration of 25 ng / mL and 25 ng / mL, respectively. The bottle was then incubated at 37°C with 5% CO2 for <1 h.

[0181] ③ Collect T cells 1*10 after 1 hour of activation. 7 Cells / groups were treated with 5 μg / mL of plasmid expressing the E1 CAR sequence and 100 μg / mL of JL transposase mRNA. The mixture was transferred to an electroporation cuvette and placed in a Lonza Nucleofactor 4D or Maxcyte electroporator. Electroporation was performed using either FI-115 or Resting T / Expand T4 program. The electroporated cell suspension was then transferred to a T75 culture flask (culture medium: AIM-V medium containing 5% SR), mixed, and incubated at 37°C with 5% CO2 for 20 min to recover. CAR-T cells were then harvested and cryopreserved. The total product preparation cycle, from cell sorting to CAR-T cell harvesting, was approximately 6 hours.

[0182] (2) Multi-round co-culture of tumor cells

[0183] Similar to Example 4, CAR-T cells (BZE2204) prepared by the 6-hour process were co-cultured with three types of tumor cells and a mixture of tumor cells. The tumor cell killing rate in each round of culture is shown in Figure 18, and the fold increase of CAR-T cells in multiple rounds of culture is shown in Figure 19. The CAR-T cells prepared by the 6-hour process exhibited comparable tumor cell cytotoxicity to BMKs and also showed higher proliferation potential.

[0184] (3) Preclinical research

[0185] Similar to Example 4, the Vehicle, MOCK-T, and high (3.3 × 10) 6 (1×10) 6(number), low (0.1×10) 6 Three doses of CAR-T cells were injected intravenously into Raji tumor mice in a single dose, and the results were observed for 4 weeks. The results showed that in the Raji tumor mouse model, the lower dose of CAR-T cells (3.3 × 10⁻⁶ cells) resulted in better outcomes. 6 -5×10 5 CAR-T cells can produce highly efficient antigen-specific killing effects (Figures 20A and 21); CAR-T cells have almost no effect on mouse body weight, indicating good safety (Figure 20B); CAR-T cells exhibit strong proliferative potential in vivo and have high pharmacokinetic (PK) values ​​(Figure 20C); the proportion of CAR-T cells in CD3-positive cells indicates (Figure 20D), and the CAR-T cell proliferation in Figure 20C is antigen-specific.

[0186] (4) Clinical trial data

[0187] CAR-T cells prepared using a 6-hour process were used in a human clinical trial (NCT06446128). This single-center, open-label exploratory study enrolled patients in two cohorts, receiving either a lymphocyte-depletion (LD) regimen (fludarabine / cyclophosphamide, dose 30 / 300, administered for 3 consecutive days) or a non-lymphocyte-depletion (NLD) regimen. Patients in the LD cohort received a single infusion of 5 × 10⁻⁶ CAR-T cells. 4 -2×10 5 Patients in the NLD cohort received CAR-T cell infusion after apheresis, with a dose of 2 × 10⁻⁶ cells per kilogram of body weight; 5 -5×10 5 Individuals per kilogram of body weight.

[0188] A total of 8 patients with recurrent large B-cell lymphoma (rrB-NHL) were included (LD cohort n=5, NLD n=3). In the LD cohort, 5 patients (4 with diffuse large B-cell lymphoma and 1 with follicular lymphoma) had a median maximum diameter product (SPD) of 657.62 mm at baseline. 2 The range is 213.28–11256.73 mm. 2 In the NLD cohort, the median maximum diameter product (SPD) of the baseline tumor volume was 2280.86 mm in 3 patients (all with diffuse large B-cell lymphoma). 2 The range is 492.28-2702.91mm. 2All patients achieved an objective response (ORR) of 100% (8 / 8) and an overall complete response (CRR) of 87.5% (7 / 8). The complete response rate (CRR) in the LD cohort was 80.0% (4 / 5), with a median follow-up of 6.8 months (range 2.3–8.6 months). PET-CT images of representative CR patients in the LD cohort are shown in Figure 22, with sustained complete response (CR) exceeding 9 months. All three patients in the NLD cohort achieved complete response (CRR) of 100% (3 / 3), with a median follow-up of 3 months (range 2.2–4.3 months). Median progression-free survival (PFS), median overall survival (OS), and median duration of response (DOR) have not yet been reached. All patients are still under close monitoring to assess the safety and efficacy.

[0189] In terms of pharmacokinetics, the three-target CAR-T cells exhibited robust proliferative capacity, requiring only 1 / 20 to 1 / 5 the dose of traditional CAR-T cells. As shown in Figure 23, in the LD cohort, the median peak plasma concentration (Cmax) of CAR-T cells was 2315 cells / μL (range: 1051–10974 cells / μL), and the time to peak concentration (Tmax) was 14 days (range: 11–14 days); CAR-T cells were still detectable in some patients after 6 months. As shown in Figure 24, in the NLD cohort, the time to peak concentration (Tmax) of CAR-T cells was also 14 days (range: 11–14 days), and the median peak plasma concentration (Cmax) was 1101 cells / μL (range: 366–4543 cells / μL).

[0190] No grade 3 or higher cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS) occurred in this study, and no dose-limiting toxicities (DLTs) were observed. In the lymphatic clearance (LD) group, all 5 patients experienced grade 1 CRS, and 2 / 5 experienced grade 1-2 ICANS; in the non-lymphatic clearance (NLD) group, 2 / 3 of patients experienced grade 1 CRS, and 1 / 3 experienced grade 2 ICANS. Grade ≥3 hematologic toxicities were observed in the LD group, including neutropenia (4 / 5), thrombocytopenia (2 / 5), and anemia (1 / 5), with 2 patients experiencing infectious events (1 of which was grade 3). In contrast, no grade ≥3 hematologic toxicities or infectious events were reported in the NLD group. The non-lymphatic clearance regimen demonstrated a significant advantage in reducing the risk of toxicity and infection.

[0191] The above description is merely a preferred embodiment of this disclosure and is not intended to limit the scope of this disclosure. Various modifications and variations can be made to this disclosure by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this disclosure should be included within the protection scope of this disclosure.

[0192] Part of the sequence in this article

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Claims

1. A multispecific antigen-binding molecule, characterized in that, The invention comprises a multispecific antibody or an antigen-binding fragment thereof, wherein the multispecific antibody contains: a first functional region targeting CD19, a second functional region targeting CD22 and a third functional region targeting BCMA, wherein the first functional region is an anti-CD19 heavy chain antibody or an antigen-binding fragment thereof, the second functional region is an anti-CD22 heavy chain antibody or an antigen-binding fragment thereof, and the third functional region is an anti-BCMA heavy chain antibody or an antigen-binding fragment thereof. The complementarity-determining region of the anti-CD19 heavy chain antibody includes: the CDR1 sequence shown in any one of SEQ ID NO:1-5, the CDR2 sequence shown in any one of SEQ ID NO:6-10, and the CDR3 sequence shown in any one of SEQ ID NO:11-15; The complementarity-determining region of the anti-CD22 heavy chain antibody includes: the CDR1 sequence shown in any one of SEQ ID NO:16-22, the CDR2 sequence shown in any one of SEQ ID NO:23-29, and the CDR3 sequence shown in any one of SEQ ID NO:30-36; The complementarity-determining region of the anti-BCMA heavy chain antibody includes: the CDR1 sequence shown in any one of SEQ ID NO:37-40, the CDR2 sequence shown in any one of SEQ ID NO:41-44, and the CDR3 sequence shown in any one of SEQ ID NO:45-48.

2. The multispecific antigen-binding molecule as described in claim 1, characterized in that, The complementarity-determining region of the anti-CD19 heavy chain antibody includes: CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:1, 6, and 11, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:2, 7, and 12, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:3, 8, and 13, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:4, 9, and 14, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:5, 10, and 15, respectively. The complementarity-determining region of the anti-CD22 heavy chain antibody comprises: CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:16, 23, and 30, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:17, 24, and 31, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:18, 25, and 32, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:19, 26, and 33, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:20, 27, and 34, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:21, 28, and 35, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:22, 29, and 36, respectively. Furthermore, the complementarity-determining region of the anti-BCMA heavy chain antibody comprises: CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:37, 41, and 45, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:38, 42, and 46, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:39, 43, and 47, respectively; or CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:40, 44, and 48, respectively.

3. The multispecific antigen-binding molecule as described in claim 1 or 2, characterized in that, The multispecific antibodies are linked in the following order: the third functional region targeting BCMA, the first functional region targeting CD19, and the second functional region targeting CD22. The complementarity-determining region of the anti-CD19 heavy chain antibody includes the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1, 6, and 11, respectively. The complementarity-determining region of the anti-CD22 heavy chain antibody includes the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:17, 24, and 31, respectively. The complementarity-determining region of the anti-BCMA heavy chain antibody includes the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:37, 41, and 45, respectively. Alternatively, the multispecific antibody may be linked in the following order: a second functional region targeting CD22, a third functional region targeting BCMA, and a first functional region targeting CD19. The complementarity-determining region of the anti-CD19 heavy chain antibody may contain the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:1, 6, and 11, respectively. The complementarity-determining region of the anti-CD22 heavy chain antibody may contain the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:17, 24, and 31, respectively. The complementarity-determining region of the anti-BCMA heavy chain antibody may contain the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:37, 41, and 45, respectively. Alternatively, the multispecific antibody may be linked in the following order: a first functional region targeting CD19, a third functional region targeting BCMA, and a second functional region targeting CD22. The complementarity-determining region of the anti-CD19 heavy chain antibody may contain the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:5, 10, and 15, respectively. The complementarity-determining region of the anti-CD22 heavy chain antibody may contain the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:17, 24, and 31, respectively. The complementarity-determining region of the anti-BCMA heavy chain antibody may contain the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:37, 41, and 45, respectively. Alternatively, the multispecific antibody may be linked in the following order: a third functional region targeting BCMA, a first functional region targeting CD19, and a second functional region targeting CD22. The complementarity-determining region of the anti-CD19 heavy chain antibody may contain CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:4, 9, and 14, respectively. The complementarity-determining region of the anti-CD22 heavy chain antibody may contain CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:18, 25, and 32, respectively. The complementarity-determining region of the anti-BCMA heavy chain antibody may contain CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:37, 41, and 45, respectively. Alternatively, the multispecific antibody may be linked in the following order: a third functional region targeting BCMA, a first functional region targeting CD19, and a second functional region targeting CD22. The complementarity-determining region of the anti-CD19 heavy chain antibody may contain CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:3, 8, and 13, respectively. The complementarity-determining region of the anti-CD22 heavy chain antibody may contain CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:17, 24, and 31, respectively. The complementarity-determining region of the anti-BCMA heavy chain antibody may contain CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:37, 41, and 45, respectively. Alternatively, the multispecific antibody may be linked in the following order: a first functional region targeting CD19, a third functional region targeting BCMA, and a second functional region targeting CD22. The complementarity-determining region of the anti-CD19 heavy chain antibody may contain the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:3, 8, and 13, respectively. The complementarity-determining region of the anti-CD22 heavy chain antibody may contain the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:17, 24, and 31, respectively. The complementarity-determining region of the anti-BCMA heavy chain antibody may contain the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:37, 41, and 45, respectively. Alternatively, the multispecific antibody may be linked in the following order: a third functional region targeting BCMA, a second functional region targeting CD22, and a first functional region targeting CD19. The complementarity-determining region of the anti-CD19 heavy chain antibody may contain CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:1, 6, and 11, respectively. The complementarity-determining region of the anti-CD22 heavy chain antibody may contain CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:18, 25, and 32, respectively. The complementarity-determining region of the anti-BCMA heavy chain antibody may contain CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:38, 42, and 46, respectively. Alternatively, the multispecific antibody may be linked in the following order: a third functional region targeting BCMA, a second functional region targeting CD22, and a first functional region targeting CD19. The complementarity-determining region of the anti-CD19 heavy chain antibody may contain CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:1, 6, and 11, respectively. The complementarity-determining region of the anti-CD22 heavy chain antibody may contain CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:18, 25, and 32, respectively. The complementarity-determining region of the anti-BCMA heavy chain antibody may contain CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:39, 43, and 47, respectively. Alternatively, the multispecific antibody may be linked in the following order: a second functional region targeting CD22, a third functional region targeting BCMA, and a first functional region targeting CD19. The complementarity-determining region of the anti-CD19 heavy chain antibody may contain the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:1, 6, and 11, respectively. The complementarity-determining region of the anti-CD22 heavy chain antibody may contain the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:17, 24, and 31, respectively. The complementarity-determining region of the anti-BCMA heavy chain antibody may contain the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:38, 42, and 46, respectively. Alternatively, the multispecific antibody may be linked in the following order: a third functional region targeting BCMA, a first functional region targeting CD19, and a second functional region targeting CD22. The complementarity-determining region of the anti-CD19 heavy chain antibody may contain CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:1, 6, and 11, respectively. The complementarity-determining region of the anti-CD22 heavy chain antibody may contain CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:18, 25, and 32, respectively. The complementarity-determining region of the anti-BCMA heavy chain antibody may contain CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:37, 41, and 45, respectively. The complementarity-determining region (CDR) of the anti-CD19 heavy chain antibody includes the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1, 6, and 11, respectively; the CDR1, CDR2, and CDR3 sequences of the anti-CD22 heavy chain antibody include the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:18, 25, and 32, respectively; and the CDR1, CDR2, and CDR3 sequences of the anti-BCMA heavy chain antibody include the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:38, 42, and 46, respectively. Alternatively, the multispecific antibody may be linked in the following order: a second functional region targeting CD22, a third functional region targeting BCMA, and a first functional region targeting CD19. The complementarity-determining region of the anti-CD19 heavy chain antibody may contain the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:1, 6, and 11, respectively. The complementarity-determining region of the anti-CD22 heavy chain antibody may contain the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:22, 29, and 36, respectively. The complementarity-determining region of the anti-BCMA heavy chain antibody may contain the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:37, 41, and 45, respectively. Alternatively, the multispecific antibody may be linked in the following order: a third functional region targeting BCMA, a first functional region targeting CD19, and a second functional region targeting CD22. The complementarity-determining region of the anti-CD19 heavy chain antibody may contain the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:1, 6, and 11, respectively. The complementarity-determining region of the anti-CD22 heavy chain antibody may contain the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:20, 27, and 34, respectively. The complementarity-determining region of the anti-BCMA heavy chain antibody may contain the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:39, 43, and 47, respectively. Alternatively, the multispecific antibody may be linked in the following order: a first functional region targeting CD19, a second functional region targeting CD22, and a third functional region targeting BCMA. The complementarity-determining region (CDDMR) of the anti-CD19 heavy chain antibody may contain the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:5, 10, and 15, respectively. The CD22 heavy chain antibody may contain the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:19, 26, and 33, respectively. The BCMA heavy chain antibody may contain the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:40, 44, and 48, respectively.

4. The multispecific antigen-binding molecule according to any one of claims 1-3, characterized in that, The anti-CD19 heavy chain antibody has the sequences shown in SEQ ID NO:49-59, the anti-CD22 heavy chain antibody has the sequences shown in any one of SEQ ID NO:60-74, and the anti-BCMA heavy chain antibody has the sequences shown in SEQ ID NO:75-82; Preferably, the first functional region, the second functional region, and the third functional region are fused together via a connector; more preferably, the multispecific antibody contains any of the sequences shown in SEQ ID NO:83-96.

5. A fusion protein comprising the multispecific antigen-binding molecule as described in any one of claims 1-4 and other polypeptides. Preferably, the other polypeptides are located at the N-terminus and / or C-terminus of the binding molecule. Preferably, the other polypeptides include polypeptides that localize binding molecules to different organelles, tags for purification or for immune responses, transmembrane proteins or their transmembrane regions, chimeric antigen receptors or components thereof, and more preferably, the fusion protein is a chimeric antigen receptor.

6. A nucleic acid molecule comprising a sequence selected from: (1) The coding sequence of the multispecific antigen-binding molecule according to any one of claims 1-4 or the fusion protein according to claim 5; (2)(1) complementary sequences.

7. A nucleic acid construct comprising the nucleic acid molecule of claim 6, preferably, the nucleic acid construct being a cloning vector, an expression vector, or an integration vector.

8. A host cell, wherein the host cell: (1) Expressing and / or secreting the multispecific antigen-binding molecule of any one of claims 1-4 or the fusion protein of claim 5; (2) Contains the nucleic acid molecule of claim 6 or the nucleic acid construct of claim 7; Preferably, the host cell is an immune effector cell, more preferably a T cell.

9. A method for producing a multispecific antigen-binding molecule according to any one of claims 1-4 or a fusion protein according to claim 5, comprising: The host cells described herein are cultured under conditions suitable for producing multispecific antigen-binding molecules or the fusion protein, and optionally, the multispecific antigen-binding molecules are purified from the culture. Nucleic acid molecules encoding the multispecific antigen-binding molecule or the fusion protein are incubated under conditions suitable for the translation of DNA or RNA in a non-cellular system (e.g., solution).

10. A pharmaceutical composition comprising the multispecific antigen-binding molecule of any one of claims 1-4, the fusion protein of claim 5, the nucleic acid molecule of claim 6, the nucleic acid construct of claim 7, or the host cell of claim 8, and pharmaceutically acceptable excipients.

11. Use of the multispecific antigen-binding molecule according to any one of claims 1-4, the fusion protein according to claim 5, the nucleic acid molecule according to claim 6, the nucleic acid construct according to claim 7, or the host cell according to claim 8 in the preparation of engineered immune cells. Preferably, the immune cells are T cells.

12. Use of the multispecific antigen-binding molecule of any one of claims 1-4, the fusion protein of claim 5, the nucleic acid molecule of claim 6, the nucleic acid construct of claim 7, or the host cell of claim 8 in the preparation of a medicament for the prevention and / or treatment of diseases or conditions related to CD19, CD22, and / or BCMA expression. Preferably, the disease or condition is cancer, such as selected from B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), multiple myeloma, follicular lymphoma, spleen, marginal zone lymphoma, mantle cell lymphoma, indolent B-cell lymphoma or Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, relapsed or refractory acute lymphoblastic leukemia (r / r ALL), relapsed or refractory diffuse large B-cell lymphoma (r / r DLBCL), and relapsed or refractory follicular lymphoma (r / r FL).

13. A method for treating or preventing diseases or conditions related to CD19, CD22, and / or BCMA expression, characterized in that, The method comprises administering to a patient in need a therapeutically effective amount of the multispecific antigen-binding molecule of any one of claims 1-4, the fusion protein of claim 5, the nucleic acid molecule of claim 6, the nucleic acid construct of claim 7, the host cell of claim 8, or the pharmaceutical composition of claim 10.

14. The method as described in claim 13, characterized in that, The method includes administering a therapeutically effective amount of CAR-T cells or a pharmaceutical composition of CAR-T cells to a patient in need, wherein the CAR-T cells express an antigen-binding domain of a chimeric antigen receptor comprising the multispecific antigen-binding molecule of any one of claims 1-4; preferably, lymphocyte depletion is not performed on the patient prior to administration of the CAR-T cells.

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