Patent Application
20250114467
The present disclosure relates to the fields of biotechnology and medicine, and particularly relates to an anti-DLL3 antibody and preparation method therefor, drug conjugate and application thereof.
Small cell lung cancer (SCLC) is the most malignant type of lung cancer, characterized by rapid progression, early metastasis, and easy recurrence. It accounts for approximately 15%-20% of new-onset lung cancer cases and is closely associated with long-term smoking. SCLC patients are primarily divided into patients in limited-stage and extensive-stage, of which patients in the extensive-stage are the majority, accounting for about 70% of SCLC patients. At present, the treatment of SCLC is primarily based on chemotherapy. The first-line chemotherapy is mainly a combination therapy based on platinum drugs, with etoposide+cisplatin or carboplatin (EP or CE regimen) being the most commonly used. In guidelines, it is recommended that both limited-stage and extensive-stage SCLC patients should receive 4-6 courses of combination therapy with cisplatin/carboplatin. The response rate for first-line treatment in limited-stage SCLC is 70%-90%, while in extensive-stage SCLC, it is 50%-60%. Currently, within one year of first-line treatment for SCLC, about 80% of patients with limited-stage and nearly all patients with extensive-stage experience relapse or progression. Therefore, the efficacy of second-line treatment significantly impacts the ultimate survival outcomes of SCLC patients. Topotecan, as a standard monotherapy for second-line treatment of SCLC, has a response rate of about 22%, with an overall survival of approximately 8 months. However, for patients with drug-resistant relapse, the response rate drops to around 4%, and the median survival is only 5 months, indicating extremely limited clinical benefit and a significant unmet clinical need. In summary, due to the high recurrence and drug resistance rates of SCLC, coupled with limited options for second-line treatment, it results in limited survival benefits, involving numerous challenges in treatment. There is an urgent need for new therapeutic methods to address these unmet clinical needs. Current research indicates that the inhibition of the Notch pathway is highly associated with the development and progression of SCLC. Therefore, its ligand, DLL3, is considered one of the most promising targets for the treatment of SCLC.
DLL3 is an atypical Notch pathway ligand identified through high-throughput sequencing. It is a single transmembrane protein composed of 619 amino acids. Its complete structure includes one DSL domain, one intracellular domain, and six epidermal growth factor-like domains (namely domains EGF1, EGF2, EGF3, EGF4, EGF5, and EGF6), making it a crucial target in the development and progression of SCLC. Immunohistochemistry reveals that this target is hardly expressed in normal tissues and is not expressed in other types of tumors. However, it is specifically and abundantly expressed in neuroendocrine tumors. Approximately 80% of SCLC tumor tissues and cancer cell surfaces present high levels of DLL3 expression. Similarly, about 85% of recurrent SCLC cases exhibit high expression of the DLL3 protein, making it a constitutive expression receptor of SCLC. The lack of DLL3 expression in normal tissues and its specific expression in neuroendocrine tumors such as SCLC making it a viable target for the development of antibodies or antibody-drug conjugates (ADCs).
Currently, various drugs targeting the DLL3 target are under development, mainly including bispecific antibodies, cell therapies, and ADCs (antibody-drug conjugates) (e.g., see CN104520324A). Rova-T, developed by Abb Vie, is the first ADC drug targeting the DLL3 target and the first targeting therapeutic drug for SCLC in clinical research. This drug utilizes DLL3 expressed on the surface of tumor cells to recognize the tumor cells and deliver the cytotoxic drug PBD into the tumor cells, achieving the effect of targeted killing of the tumor cells. However, in phase III clinical trials, due to the serious toxic side effects of the cytotoxic agent PBD, which results in the intolerance of the patients, leading to insufficient efficacy, the development of this drug has been terminated.
The technical problem addressed by the present disclosure is the inadequacies of existing anti-DLL3 antibodies and the current clinical situation that ADC drugs targeting DLL3 have not been successfully developed. To address these issues, the present disclosure provides a novel anti-DLL3 antibody, an ADC targeting DLL3, an intermediate thereof, a preparation method therefor, and a use thereof. Addressing the deficiency in prior art of lacking antibody-drug conjugates targeting DLL3, the antibody-drug conjugate described in the present disclosure can effectively treat patients with tumors exhibiting neuroendocrine characteristics, including SCLC. The present disclosure primarily employs the following technical means to solve the aforementioned technical problems.
The present disclosure provides an anti-DLL3 antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein
In the present application, the term “amino acid mutation” in a context like “having 3, 2, or 1 amino acid insertions, deletions, or substitutions” refers to an amino acid mutation in a varied sequence relative to the original amino acid sequence, which includes insertion, deletion, or substitution of amino acids based on the original amino acid sequence. An exemplary explanation is that the mutations in CDR can involve mutations of 3, 2, or 1 amino acid(s), and the same or different number of amino acid residues can optionally be selected for mutation between these CDRs. For example, there could be a mutation of one amino acid in CDR1, with no amino acid mutations in CDR2 and CDR3.
In the present application, the mutations may include those currently known to those skilled in the art, such as mutations that might be made to antibodies during their production or application. For instance, mutations may be made at sites of potential post-translational modifications (PTMs), especially in the CDR regions. These include mutations related to antibody aggregation, susceptible to asparagine deamidation (at sites like NG, NS, NH, etc.), susceptible to aspartic acid isomerization (at DG, DP sites), susceptible to N-glycosylation (at N−{P}S/T sites), and susceptible to oxidation, and other related mutations.
Preferably, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 comprised in the VH are respectively as shown in SEQ ID NO: 9, 10, and 11; or the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 comprised in the VH are respectively as shown in SEQ ID NO: 19, 20, and 21; or the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 comprised in the VH are respectively as shown in SEQ ID NO: 29, 30, and 31; or the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 comprised in the VH are respectively as shown in SEQ ID NO: 39, 40, and 41; or the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 comprised in the VH are respectively as shown in SEQ ID NO: 59, 60, and 61; or the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 comprised in the VH are respectively as shown in SEQ ID NO: 69, 70, and 71; or the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 comprised in the VH are respectively as shown in SEQ ID NO: 79, 80, and 81; or the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 comprised in the VH are respectively as shown in SEQ ID NO: 89, 90, and 91; or the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 comprised in the VH are respectively as shown in SEQ ID NO: 99, 100, and 101; or the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 comprised in the VH are respectively as shown in SEQ ID NO: 89, 110, and 111; or the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 comprised in the VH are respectively as shown in SEQ ID NO: 63, 83, and 93.
Preferably, the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 comprised in the VL are respectively as shown in SEQ ID NO: 12, GAS, and SEQ ID NO: 14; or the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 comprised in the VL are respectively as shown in SEQ ID NO: 12, GAS, and SEQ ID NO: 24; or the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 comprised in the VL are respectively as shown in SEQ ID NO: 32, GAT, and SEQ ID NO: 34; or the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 comprised in the VL are respectively as shown in SEQ ID NO: 42, TTS, and SEQ ID NO: 44; or the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 comprised in the VL are respectively as shown in SEQ ID NO: 62, NAK, and SEQ ID NO: 64; or the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 comprised in the VL are respectively as shown in SEQ ID NO: 72, NAK, and SEQ ID NO: 74; or the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 comprised in the VL are respectively as shown in SEQ ID NO: 82, YTS, and SEQ ID NO: 84; or the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 comprised in the VL are respectively as shown in SEQ ID NO: 92, RAN, and SEQ ID NO: 94; or the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 comprised in the VL are respectively as shown in SEQ ID NO: 102, WAS, and SEQ ID NO: 104; or the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 comprised in the VL are respectively as shown in SEQ ID NO: 112, FTS, and SEQ ID NO: 114; or the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 comprised in the VL are respectively as shown in SEQ ID NO: 103, NAN, and 113.
In one preferred embodiment, in the anti-DLL3 antibody, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 comprised in the VH are respectively as shown in SEQ ID NO: 9, 10, and 11; and the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 comprised in the VL are respectively as shown in SEQ ID NO: 12, GAS, and SEQ ID NO: 14. In one preferred embodiment, in the anti-DLL3 antibody, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 comprised in the VH are respectively as shown in SEQ ID NO: 19, 20, and 21; and the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 comprised in the VL are respectively as shown in SEQ ID NO: 12, GAS, and SEQ ID NO: 24. In one preferred embodiment, in the anti-DLL3 antibody, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 comprised in the VH are respectively as shown in SEQ ID NO: 29, 30, and 31; and the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 comprised in the VL are respectively as shown in SEQ ID NO: 32, GAT, and SEQ ID NO: 34. In one preferred embodiment, in the anti-DLL3 antibody, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 comprised in the VH are respectively as shown in SEQ ID NO: 39, 40, and 41; and the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 comprised in the VL are respectively as shown in SEQ ID NO: 42, TTS, and SEQ ID NO: 44. In one preferred embodiment, in the anti-DLL3 antibody, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 comprised in the VH are respectively as shown in SEQ ID NO: 59, 60, and 61; and the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 comprised in the VL are respectively as shown in SEQ ID NO: 62, NAK, and SEQ ID NO: 64. In one preferred embodiment, in the anti-DLL3 antibody, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 comprised in the VH are respectively as shown in SEQ ID NO: 69, 70, and 71; and the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 comprised in the VL are respectively as shown in SEQ ID NO: 72, NAK, and SEQ ID NO: 74. In one preferred embodiment, in the anti-DLL3 antibody, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 comprised in the VH are respectively as shown in SEQ ID NO: 79, 80, and 81; and the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 comprised in the VL are respectively as shown in SEQ ID NO: 82, YTS, and SEQ ID NO: 84. In one preferred embodiment, in the anti-DLL3 antibody, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 comprised in the VH are respectively as shown in SEQ ID NO: 89, 90, and 91; and the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 comprised in the VL are respectively as shown in SEQ ID NO: 92, RAN, and SEQ ID NO: 94. In one preferred embodiment, in the anti-DLL3 antibody, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 comprised in the VH are respectively as shown in SEQ ID NO: 99, 100, and 101; and the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 comprised in the VL are respectively as shown in SEQ ID NO: 102, WAS, and SEQ ID NO: 104. In one preferred embodiment, in the anti-DLL3 antibody, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 comprised in the VH are respectively as shown in SEQ ID NO: 89, 110, and 111; and the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 comprised in the VL are respectively as shown in SEQ ID NO: 112, FTS, and SEQ ID NO: 114. In one preferred embodiment, in the anti-DLL3 antibody, the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 comprised in the VH are respectively as shown in SEQ ID NO: 63, 83, and 93; and the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 comprised in the VL are respectively as shown in SEQ ID NO: 103, NAN, and 113. Preferably, in the anti-DLL3 antibody, the heavy chain variable region (VH) further comprises a heavy chain variable region framework region (VH FWR), wherein the VH FWR is the heavy chain variable region framework region of a human or murine antibody. In one preferred embodiment of the present disclosure, the VH may comprise the amino acid sequence as shown in SEQ ID NO: 15, 25, 35, 45, 65, 75, 85, 95, 105, 13, or 22, or mutations thereof.
Preferably, in the anti-DLL3 antibody, the light chain variable region (VL) further comprises a light chain variable region framework region (VL FWR), wherein the VL FWR is the light chain variable region framework region of a human or murine antibody. In one preferred embodiment of the present disclosure, the VL may comprise the amino acid sequence as shown in SEQ ID NO: 16, 26, 36, 46, 66, 76, 86, 96, 106, 33, or 23, or mutations thereof.
In one preferred embodiment, the VH comprises the amino acid sequence as shown in SEQ ID NO: 15 or mutations thereof, and the VL comprises the amino acid sequence as shown in SEQ ID NO: 16 or mutations thereof. In one preferred embodiment, the VH comprises the amino acid sequence as shown in SEQ ID NO: 25 or mutations thereof, and the VL comprises the amino acid sequence as shown in SEQ ID NO: 26 or mutations thereof. In one preferred embodiment, the VH comprises the amino acid sequence as shown in SEQ ID NO: 35 or mutations thereof, and the VL comprises the amino acid sequence as shown in SEQ ID NO: 36 or mutations thereof. In one preferred embodiment, the VH comprises the amino acid sequence as shown in SEQ ID NO: 45 or mutations thereof, and the VL comprises the amino acid sequence as shown in SEQ ID NO: 46 or mutations thereof. In one preferred embodiment, the VH comprises the amino acid sequence as shown in SEQ ID NO: 65 or mutations thereof, and the VL comprises the amino acid sequence as shown in SEQ ID NO: 66 or mutations thereof. In one preferred embodiment, the VH comprises the amino acid sequence as shown in SEQ ID NO: 75 or mutations thereof, and the VL comprises the amino acid sequence as shown in SEQ ID NO: 76 or mutations thereof. In one preferred embodiment, the VH comprises the amino acid sequence as shown in SEQ ID NO: 85 or mutations thereof, and the VL comprises the amino acid sequence as shown in SEQ ID NO: 86 or mutations thereof. In one preferred embodiment, the VH comprises the amino acid sequence as shown in SEQ ID NO: 95 or mutations thereof, and the VL comprises the amino acid sequence as shown in SEQ ID NO: 96 or mutations thereof. In one preferred embodiment, the VH comprises the amino acid sequence as shown in SEQ ID NO: 105 or mutations thereof, and the VL comprises the amino acid sequence as shown in SEQ ID NO: 106 or mutations thereof. In one preferred embodiment, the VH comprises the amino acid sequence as shown in SEQ ID NO: 13 or mutations thereof, and the VL comprises the amino acid sequence as shown in SEQ ID NO: 33 or mutations thereof. In one preferred embodiment, the VH comprises the amino acid sequence as shown in SEQ ID NO: 22 or mutations thereof, and the VL comprises the amino acid sequence as shown in SEQ ID NO: 23 or mutations thereof.
The mutations involve the deletion, substitution, or addition of one or more amino acid residues in the amino acid sequence of VH and/or VL. The amino acid sequence of the mutation has at least 85% sequence identity with the amino acid sequence of VH and/or VL and maintains or improves the binding ability of the antibody to DLL3. The at least 85% sequence identity is preferably at least 90% sequence identity, more preferably at least 95%, 96%, 97%, 98% sequence identity, and most preferably at least 99% sequence identity.
In the present disclosure, the amino acid sequences of the CDRs are all presented according to the Kabat definition rules (the sequences in this application are also presented according to the Kabat definition rules). However, it is well known to those skilled in the art that there are various methods to define the CDRs of an antibody in this art, such as the Kabat definition rules based on sequence variability (see, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, National Institutes of Health, Bethesda, Maryland (1991)) and the Chothia definition rules based on the structural loop region location (see J Mol Biol 273:927-48, 1997). It should be understood by those skilled in the art that, unless otherwise specified, the terms “CDR” and “complementarity-determining region” for a given antibody or its region (such as the variable region) are to be understood as encompassing the complementarity-determining region defined by any of the known methods described in the present disclosure. Although the scope of protection requested in the present disclosure is based on sequences shown according to the Kabat definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of the present disclosure.
Preferably, the anti-DLL3 antibody is selected from the group consisting of a full-length antibody, Fab, Fab′, F(ab′)2, Fv, preferably scFv, bispecific antibody, multispecific antibody, heavy chain antibody, or single domain antibody.
In one preferred embodiment, the anti-DLL3 antibody is a full-length antibody, comprising a heavy chain and a light chain; a heavy chain constant region comprised in the heavy chain is preferably derived from human or murine antibody heavy chain constant region; a light chain constant region comprised in the light chain is preferably derived from human or murine antibody light chain constant region. Preferably, the human light chain constant region is a human κ or λ light chain constant region; the human heavy chain constant region is a human IgG1, IgG2, IgG3, or IgG4.
In one preferred embodiment, the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 17, 27, 37, 47, 67, 77, 87, 97, 107, 43, or 73, or mutations thereof, and/or the light chain comprises the amino acid sequence as shown in SEQ ID NO: 18, 28, 38, 48, 68, 78, 88, 98, 108, 53, or 109, or mutations thereof.
In one preferred embodiment, in the full-length antibody, the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 17 or mutations thereof, and the light chain comprises the amino acid sequence as shown in SEQ ID NO: 18 or mutations thereof. In one preferred embodiment, the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 27 or mutations thereof, and the light chain comprises the amino acid sequence as shown in SEQ ID NO: 28 or mutations thereof. In one preferred embodiment, the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 37 or mutations thereof, and the light chain comprises the amino acid sequence as shown in SEQ ID NO: 38 or mutations thereof. In one preferred embodiment, the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 47 or mutations thereof, and the light chain comprises the amino acid sequence as shown in SEQ ID NO: 48 or mutations thereof. In one preferred embodiment, the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 67 or mutations thereof, and the light chain comprises the amino acid sequence as shown in SEQ ID NO: 68 or mutations thereof. In one preferred embodiment, the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 77 or mutations thereof, and the light chain comprises the amino acid sequence as shown in SEQ ID NO: 78 or mutations thereof. In one preferred embodiment, the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 87 or mutations thereof, and the light chain comprises the amino acid sequence as shown in SEQ ID NO: 88 or mutations thereof. In one preferred embodiment, the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 97 or mutations thereof, and the light chain comprises the amino acid sequence as shown in SEQ ID NO: 98 or mutations thereof. In one preferred embodiment, the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 107 or mutations thereof, and the light chain comprises the amino acid sequence as shown in SEQ ID NO: 108 or mutations thereof. In one preferred embodiment, the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 43 or mutations thereof, and the light chain comprises the amino acid sequence as shown in SEQ ID NO: 53 or mutations thereof. In one preferred embodiment, the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 73 or mutations thereof, and the light chain comprises the amino acid sequence as shown in SEQ ID NO: 109 or mutations thereof.
The mutations involve the deletion, substitution, or addition of one or more amino acid residues in the amino acid sequence of the heavy chain and/or the light chain. The amino acid sequence of the mutation has at least 85% sequence identity with the amino acid sequence of the heavy chain and/or the light chain and maintains or improves the binding ability of the antibody to DLL3. The at least 85% sequence identity is preferably at least 90% sequence identity, more preferably at least 95%, 96%, 97%, or 98% sequence identity, and most preferably at least 99% sequence identity.
Another aspect of the present disclosure further provides an anti-DLL3 antibody, which competitively binds to the DLL3 protein with the anti-DLL3 antibody described above. Another aspect of the present disclosure provides an isolated nucleic acid encoding the anti-DLL3 antibody as described above.
Another aspect of the present disclosure provides a recombinant expression vector comprising the isolated nucleic acid as described above. Preferably, the expression vector comprises a eukaryotic cell expression vector and/or a prokaryotic cell expression vector.
Another aspect of the present disclosure provides a transformant comprising the recombinant expression vector as described above. Preferably, the host cell of the transformant is a prokaryotic and/or a eukaryotic cell, wherein the prokaryotic cell is preferably E. coli cell such as TG1 or BL21 cell, and the eukaryotic cell is preferably HEK293 cell or CHO cell.
Another aspect of the present disclosure provides an antibody-drug conjugate (ADC), with the general structure formula: Ab-(L3-L2-L1-D)m;
In (L)p, “L” independently represents one or more of phenylalanine residue, alanine residue, glycine residue, glutamic acid residue, aspartic acid residue, cysteine residue, glutamine residue, histidine residue, isoleucine residue, leucine residue, lysine residue, methionine residue, proline residue, serine residue, threonine residue, tryptophan residue, tyrosine residue, and valine residue; “p” is from 2 to 4.
R1 is C1-C6 alkyl substituted by one or more —NR1-1R1-2, C1-C6 alkyl substituted by one or more R1-3S(O)2—, C1-C6 alkyl, C3-C10 cycloalkyl, C6-C14 aryl, or 5- to 14-membered heteroaryl; the heteroatom in the 5- to 14-membered heteroaryl is selected from one or more of N, O, and S, and the number of heteroatoms is 1, 2, 3, or 4; the R1-1, R1-2, and R1-3 are each independently C1-C6 alkyl;
In one preferred embodiment, the anti-DLL3 antibody binds to one of the following antigenic epitopes in the DLL3 protein: DSL domain, N-terminal, EGF2 domain, and EGF3-EGF6 domains.
In one preferred embodiment, the anti-DLL3 antibody is preferably the anti-DLL3 antibody as described above.
In one preferred embodiment, the anti-DLL3 antibody binds to the antigenic epitope of the EGF2 domain of the DLL3 protein.
In one preferred embodiment, the anti-DLL3 antibody comprises a VH with an amino acid sequence as shown in SEQ ID NO: 15 and a VL with an amino acid sequence as shown in SEQ ID NO: 16, or comprises a VH with an amino acid sequence as shown in SEQ ID NO: 25 and a VL with an amino acid sequence as shown in SEQ ID NO: 26.
In one preferred embodiment, the anti-DLL3 antibody comprises a light chain with an amino acid sequence as shown in SEQ ID NO: 1 and a heavy chain with an amino acid sequence as shown in SEQ ID NO: 2, or comprises a light chain with an amino acid sequence as shown in SEQ ID NO: 3 and a heavy chain with an amino acid sequence as shown in SEQ ID NO: 4.
In one preferred embodiment, the L is one or more of phenylalanine residue, alanine residue, glycine residue, isoleucine residue, leucine residue, proline residue, and valine residue; preferably one or more of phenylalanine residue, alanine residue, glycine residue, and valine residue.
In one preferred embodiment, the L is valine residue and/or phenylalanine residue; more herein refers to two or three; the p is 2. Preferably, the (L)p is
wherein the g-terminal is connected to the c-terminal of L2 through a carbonyl group.
In one preferred embodiment, the R1 is C1-C6 alkyl substituted by one or more —NR1-1R1-2, C1-C6alkyl substituted by one or more R1-3S(O)2—, or C1-C6 alkyl; the R1-1, R1-2, and R1-3 are each independently C1-C4 alkyl.
In one preferred embodiment, when the R1 is C1-C6 alkyl substituted by one or more —NR1-1R1-2, the C1-C6 alkyl is C1-C4 alkyl, preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, further preferably ethyl; more herein refers to two or three. Preferably, the R1-1 and R1-2 are each independently C1-C4 alkyl, preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, further preferably methyl. Preferably, the —NR1-1R1-2 is —N(CH3)2. Further preferably, when the R1 is C1-C6 alkyl substituted by one —NR1-1R1-2, the C1-C6 alkyl substituted by one —NR1-1R1-2 is
In one preferred embodiment, when the R1 is C1-C6 alkyl substituted by one or more R1-3S(O)2—, the C1-C6 alkyl is C1-C4 alkyl, preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, further preferably ethyl; more herein refers to two or three. Preferably, the R1-3 is C1-C4 alkyl, preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, further preferably methyl. Preferably, when the R1 is C1-C6 alkyl substituted by one R1-3S(O)2—, the C1-C6 alkyl substituted by one R1—S(O)2— is
In one preferred embodiment, when the R1 is C1-C6 alkyl, the C1-C6 alkyl is preferably C1-C4 alkyl, further preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, further preferably methyl.
In one preferred embodiment, when the structure of L1 is as shown in formula I, the L2 is preferably
the L3 is preferably
In one preferred embodiment, when the structure of L1 is as shown in formula II, the L2 is preferably
the L3 is preferably
In one preferred embodiment of the present disclosure, when the structure of L1 is as shown in formula III, the L2 is preferably
the L3 is preferably
In one preferred embodiment of the present disclosure, when the structure of L1 is as shown in formula IV, the L2 is preferably
the L3 is preferably
In a preferred embodiment, the structure of L1 is preferably as shown in formula I or III.
In a preferred embodiment, the formula III is preferably
In one preferred embodiment, the formula III is further preferably
In one preferred embodiment, the b-terminal of L3 is preferably connected to the sulfhydryl group of the antibody in the form of a thioether bond. Taking
as an example, the connecting form of
with the cysteine residue in the antibody is
In one preferred embodiment, the m is an integer varying from 2 to 8, such as 2, 3, 4, 5, 6, 7, or 8, or a non-integer, such as 7.68, 7.53, 4.43, 7.12, 6.92, 7.43, 7.23, 6.83, 7.32, 7.56, 7.54, 7.47, 5.82, 6.78, 2.28, 6.32, 7.45, 7.65, 7.64, 7.36, 7.75, 7.80, 7.77, or 7.76.
Preferably, the antibody-drug conjugate is any one of the following compounds:
wherein Ab is the anti-DLL3 antibody as described above, and m is from 2 to 8, preferably 7.68, 7.53, 4.43, 7.12, 6.92, 7.43, 7.23, 6.83, 7.32, 7.56, 7.54, 7.47, 5.82, 6.78, 2.28, 6.32, 7.45, 7.65, 7.64, 7.36, 7.75, 7.80, 7.77, or 7.76.
In one preferred embodiment, the antibody-drug conjugate is any one of the following compounds.
wherein Ab is the anti-DLL3 antibody comprising a VH with an amino acid sequence as shown in SEQ ID NO: 25 and a VL with an amino acid sequence as shown in SEQ ID NO: 26, preferably comprising a heavy chain with an amino acid sequence as shown in SEQ ID NO: 27 and a light chain with an amino acid sequence as shown in SEQ ID NO: 28.
In one preferred embodiment, the antibody-drug conjugate is any one of the following compounds:
wherein Ab is the anti-DLL3 antibody comprising a VH with an amino acid sequence as shown in SEQ ID NO: 15 and a VL with an amino acid sequence as shown in SEQ ID NO: 16, preferably comprising a heavy chain with an amino acid sequence as shown in SEQ ID NO: 17 and a light chain with an amino acid sequence as shown in SEQ ID NO: 18.
In one preferred embodiment, the antibody-drug conjugate is the following compound:
wherein Ab is the anti-DLL3 antibody comprising a heavy chain with an amino acid sequence as shown in SEQ ID NO: 2 and a light chain with an amino acid sequence as shown in SEQ ID NO: 1.
In one preferred embodiment, the antibody-drug conjugate is the following compound:
wherein Ab is the anti-DLL3 antibody comprising a heavy chain with an amino acid sequence as shown in SEQ ID NO: 4 and a light chain with an amino acid sequence as shown in SEQ ID NO: 3.
In one preferred embodiment, the antibody-drug conjugate is the following compound:
wherein Ab is the anti-DLL3 antibody comprising a VH with an amino acid sequence as shown in SEQ ID NO: 22 and a VL with an amino acid sequence as shown in SEQ ID NO: 23, preferably comprising a heavy chain with an amino acid sequence as shown in SEQ ID NO: 73 and a light chain with an amino acid sequence as shown in SEQ ID NO: 109.
In one preferred embodiment, the antibody-drug conjugate is the following compound:
wherein Ab is the anti-DLL3 antibody comprising a VH with an amino acid sequence as shown in SEQ ID NO: 45 and a VL with an amino acid sequence as shown in SEQ ID NO: 46, preferably comprising a heavy chain with an amino acid sequence as shown in SEQ ID NO: 47 and a light chain with an amino acid sequence as shown in SEQ ID NO: 48.
Another aspect of the present disclosure further provides a chimeric antigen receptor comprising the anti-DLL3 antibody as described above.
Another aspect of the present disclosure further provides a genetically modified cell comprising the chimeric antigen receptor as described above. Preferably, the genetically modified cell is a eukaryotic cell, preferably an isolated human cell; more preferably an immune cell such as a T cell, or NK cell.
Another aspect of the present disclosure further provides a preparation method for the anti-DLL3 antibody, the preparation method comprising the following steps: cultivating the transformant as described above, and obtaining the anti-DLL3 antibody from the culture.
Another aspect of the present disclosure further provides a pharmaceutical composition comprising the anti-DLL3 antibody as described above, the antibody-drug conjugate as described above, the chimeric antigen receptor as described above, and/or the genetically modified cell as described above. Preferably, the pharmaceutical composition is in a liquid, gas, solid, or semi-solid dosage form, and/or, the pharmaceutical composition can be administered orally, by injection, nasally, transdermally, or via mucosal routes. More preferably, the pharmaceutical composition further comprises a combination therapeutic agent, and the combination therapeutic agent comprises chemotherapeutic agents, radiation therapy agents, immunosuppressants, and/or cytotoxic drugs.
Another aspect of the present disclosure further provides a use of the anti-DLL3 antibody, the antibody-drug conjugate, the chimeric antigen receptor, the genetically modified cell, and/or the pharmaceutical composition as described above in the preparation of a medicament, a kit, and/or a drug administration device for the treatment and/or prevention of diseases related to abnormal expression of DLL3; alternatively, a use of the anti-DLL3 antibody, the antibody-drug conjugate, the chimeric antigen receptor, the genetically modified cell, and/or the pharmaceutical composition as described above in the treatment and/or prevention of diseases related to abnormal expression of DLL3. The diseases related to abnormal expression of DLL3 preferably refer to tumors, the tumor is preferably cancer, and the cancer is preferably endocrine tumors such as neuroendocrine tumors, prostate cancer, pancreatic cancer, colorectal cancer, further preferably small-cell lung cancer.
Another aspect of the present disclosure also provides a kit comprising the anti-DLL3 antibody, the antibody-drug conjugate, the chimeric antigen receptor, the genetically modified cell, and/or the pharmaceutical composition as described above, and optionally, an instruction manual.
Another aspect of the present disclosure also provides a drug administration device comprising: (1) an infusion module for administering the aforementioned pharmaceutical composition to a subject in need thereof, and (2) an optional pharmacodynamic monitoring module.
Another aspect of the present disclosure also provides a method for detecting DLL3, the method comprising the step of using the anti-DLL3 antibody as described above for detection. Preferably, the method is for non-diagnostic and/or non-therapeutic purposes.
Another aspect of the present disclosure also provides a method for diagnosing, preventing, and/or treating diseases related to abnormal expression of DLL3, the method comprising administering to a subject in need thereof the anti-DLL3 antibody, the antibody-drug conjugate, the genetically modified cells, and/or the pharmaceutical composition as described above. The disease related to abnormal expression of DLL3 is preferably tumor; the tumor is preferably cancer; the cancer is preferably neuroendocrine tumors, more preferably small cell lung cancer.
The present disclosure also provides an antibody group (comprising molecules made up of or comprising antibody fragments or variants), wherein members of the group correspond to one, two, three, four, five, or more different antibodies of the present disclosure [for example, full antibodies, Fab, F(ab)2 fragments, scFv, etc.].
Another aspect of the present disclosure also provides an antibody-drug conjugate with the general structure formula Ab-(L5-L4-L3-L2-L1-D)m;
In (L)p, “L” independently represents one or more of phenylalanine residue, alanine residue, glycine residue, glutamic acid residue, aspartic acid residue, cysteine residue, glutamine residue, histidine residue, isoleucine residue, leucine residue, lysine residue, methionine residue, proline residue, serine residue, threonine residue, tryptophan residue, tyrosine residue, and valine residue; “p” is from 2 to 4.
R1 is C1-C6 alkyl substituted by one or more —NR1-1R1-2, C1-C6 alkyl substituted by one or more R1-3S(O)2—, C1-C6 alkyl, C3-C10 cycloalkyl, C6-C14 aryl, or 5- to 14-membered heteroaryl; the heteroatom in the 5- to 14-membered heteroaryl is selected from one or more of N, O, and S, and the number of heteroatoms is 1, 2, 3, or 4; the R1-1, R1-2, and R1-3 are each independently
In a preferred embodiment, D is a cytotoxic drug comprising hydroxyl, thiol, or amino groups, such as a microtubule inhibitor and/or a topoisomerase inhibitor.
In one preferred embodiment, the L is preferably one or more of phenylalanine residue, alanine residue, glycine residue, isoleucine residue, leucine residue, proline residue, and valine residue; preferably one or more of phenylalanine residue, alanine residue, glycine residue, and valine residue; further preferably, the L is the valine residue and/or the alanine residue; the “more” means two or three; p is 2.
In one preferred embodiment, the R1 is C1-C6 alkyl substituted by one or more —NR1-1R1-2, C1-C6 alkyl substituted by one or more R1-3S(O)2—, or C1-C6 alkyl; the R1-1, R1-2, and R1-3 are each independently C1-C4 alkyl.
In one preferred embodiment, when L1 has a structure as shown in formula I, the L2 is preferably
In one preferred embodiment, when L1 has a structure as shown in formula II, the L2 is preferably
In one preferred embodiment, when L1 has a structure as shown in formula III, the L2 is preferably
In one preferred embodiment, when L1 has a structure as shown in formula IV, the L2 is preferably
In one preferred embodiment, the n is independently 8, 9, 10, 11, and 12.
In one preferred embodiment, the m is an integer or non-integer from 2 to 8.
In one preferred embodiment, the L3 is
In one preferred embodiment, the L4 is selected from N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 4-iodoacetylaminobenzoate (SIAB), N-succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC), 6-maleimidocaproyl (MC), maleimidopropionic acid (MP), valine-citrulline (VC), alanine-phenylalanine (ala-phe), para-aminobenzylcarbamate (PAB), and MC-VC-PAB.
In one preferred embodiment, in the L5, when X1 is a hydrogen atom, X2 is an alkyl group, and m is 1, the compound of formula V is S-(3-carbopropyl) thioacetate.
In one preferred embodiment, D is one or more of maytansinoid derivatives, dolastatin 10 derivatives, doxorubicin analogs, or camptothecin analogs.
The maytansinoid derivatives are, for example, DM1, DM3, and DM4.
The dolastatin 10 derivatives are, for example, MMAE and MMAF.
The doxorubicin analogs are, for example, doxorubicin and camsirubicin.
More preferably, D is a camptothecin analog, further preferably a compound of formula Va or Vb:
In one preferred embodiment of the present disclosure, the (L)p is
wherein g-terminal is connected to c-terminal of the L2 through a carbonyl group.
In one preferred embodiment, the formula III is
In one preferred embodiment, the antibody-drug conjugate is any one of the following compounds:
The anti-DLL3 antibody comprises a light chain with an amino acid sequence as shown in SEQ ID NO: 1 and a heavy chain with an amino acid sequence as shown in SEQ ID NO: 2, or comprises a light chain with an amino acid sequence as shown in SEQ ID NO: 3 and a heavy chain with an amino acid sequence as shown in SEQ ID NO: 4;
Preferably, the antibody-drug conjugate is any one of the following compounds:
wherein Ab is the anti-DLL3 antibody comprising a heavy chain with an amino acid sequence as shown in SEQ ID NO: 2 and a light chain with an amino acid sequence as shown in SEQ ID NO: 1, or
wherein Ab is the anti-DLL3 antibody comprising a heavy chain with an amino acid sequence as shown in SEQ ID NO: 4 and a light chain with an amino acid sequence as shown in SEQ ID NO: 3.
Another aspect of the present disclosure also provides a preparation method for the antibody-drug conjugate as described herein, the preparation method comprising the following steps: adding an excess of linker-drug conjugate dissolved in DMSO into a buffer solution comprising the anti-DLL3 antibody, thereby obtaining the antibody-drug conjugate.
Another aspect of the present disclosure also provides a pharmaceutical composition comprising the antibody-drug conjugate as described herein.
Preferably, the pharmaceutical composition is in a liquid, gas, solid, or semi-solid dosage form, and/or, the pharmaceutical composition can be administered orally, by injection, nasally, transdermally, or via mucosal routes.
More preferably, the pharmaceutical composition further comprises a combination therapeutic agent comprising chemotherapeutic agents, radiation therapy agents, immunosuppressants, and/or cytotoxic drugs.
Another aspect of the present disclosure also provides a use of the antibody-drug conjugate and/or the pharmaceutical composition as described herein in the preparation of a medicament, a kit, and/or a drug administration device for the treatment and/or prevention of diseases related to abnormal expression of DLL3;
Another aspect of the present disclosure also provides a drug administration device, wherein the drug administration device comprises: (1) an infusion module for administering the antibody-drug conjugate and/or the pharmaceutical composition as described herein to a subject in need thereof, and (2) an optional pharmacodynamic monitoring module.
Another aspect of the present disclosure also provides a method for detecting DLL3, wherein the method for detecting DLL3 comprises the step of using the antibody-drug conjugate as described herein for detection. Preferably, the method for detecting DLL3 is for non-diagnostic and/or non-therapeutic purposes.
Another aspect of the present disclosure also provides a method for diagnosing, preventing, and/or treating diseases related to abnormal expression of DLL3, the method comprising administering to a subject in need thereof the antibody-drug conjugate and/or the pharmaceutical composition as described herein.
In the present disclosure, the conditions and operations of the conjugating reaction can be conventional conditions and operations for the conjugating reaction in the art.
In the present disclosure, m represents the molar ratio of cytotoxic drug molecules to Ab (also known as Drug-Antibody Ratio, DAR), which can be an integer or a decimal. Preferably, it is understood as the average of molar ratio of drug molecules to monoclonal antibody molecules in the antibody-drug conjugate obtained after conjugation of the monoclonal antibody molecules with the cytotoxic drugs. The ratio can generally be determined by methods such as Hydrophobic-Interaction Chromatography (HIC), Polyacrylamide-SDS Gel Electrophoresis (SDS-PAGE), and Liquid Chromatography-Mass Spectrometry (LC-MS).
In the present disclosure, the term “C1-C6 alkyl” by itself or in combination with others refers to saturated straight-chain or branched alkyl groups comprising 1 to 6 carbon atoms, particularly 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl. The term “C1-C6 alkyl” is preferably methyl or ethyl.
The antibodies of the present disclosure can be prepared using techniques well known in the art, such as hybridoma methods, recombinant DNA techniques, phage display techniques, synthetic techniques, or combinations thereof, or other techniques known in the art.
In the present disclosure, the terms “selectivity” or “specificity” refer to the fact that the disclosed antagonists do not show significant binding to substances other than DLL3, except in those special cases where additional antagonists are incorporated to confer an additional specificity, different from the DLL3-specific binding part (e.g., bispecific or bifunctional molecules, where the molecule is designed to bind or exert two functions, at least one of which involves specific binding to DLL3).
The term “antibody molecule” or “antibody” as described herein refers to immunoglobulin molecules and immunologically active parts of immunoglobulin molecules, that is, molecules comprising antigen-binding sites with immunospecific binding to antigens. Therefore, the term antibody encompasses not only complete antibody molecules but also fragments of the antibodies as well as variants of the antibodies and the antibody fragments (including derivatives). The term antibody molecule as described herein includes, but is not limited to, single-chain Fv (scFv), Fab fragments, Fab′ fragments, F(ab′)2, disulfide-linked Fv (sdFv), Fv, as well as complete or full-length antibodies. The term “single-chain Fv” or “scFv” refers to a peptide consisting of an antibody's VL (Variable Light) domain linked to the VH (Variable Heavy) domain of the antibody. Antibodies that immunospecifically bind to DLL3 may cross-react with other antigens. Preferably, antibodies that immunospecifically bind to DLL3 do not cross-react with other antigens. Antibodies that immunospecifically bind to DLL3 can be identified, for example, through immunoassays or other methods known to those skilled in the art. A “complete antibody” or “full-length antibody” refers to a protein consisting of two heavy chains (H) and two light chains (L) connected by disulfide bonds. The protein comprises: (1) for the heavy chains, a heavy chain variable region (abbreviated as “VH”) and a heavy chain constant region comprising three domains CH1, CH2, and CH3; and (2) for the light chains, a light chain variable region (abbreviated as “VL”) and a light chain constant region comprising one domain, CL. The antibodies of the present disclosure include, but are not limited to, monoclonal, multispecific, human or chimeric antibodies, single-chain antibodies, Fab fragments, F(ab′) fragments, anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against the antibodies of the present disclosure), and epitope-binding fragments of any of the aforementioned antibodies. The immunoglobulin molecules of the present disclosure can be of any type of immunoglobulin (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In the present disclosure, the antibody can be either a monoclonal antibody or a polyclonal antibody, with the monoclonal antibody preferably being a murine anti-human monoclonal antibody. The antibodies of the present disclosure can be ultra-humanized antibodies or double antibodies.
In the present disclosure, the term “heavy chain antibody” refers to an antibody that comprises only one variable domain of the heavy chain (VHH) and two conventional domains CH2 and CH3, also known as HCAbs.
A “single domain antibody,” also known as a “nanobody,” refers to the VHH structure cloned from heavy chain antibodies, representing the smallest known unit capable of binding to a target antigen.
In the present disclosure, an amino acid sequence described as having “more than 90%, 95%, 98%, or 99% identity” is obtained by insertion, deletion, or substitution of the amino acid sequences shown in the previous sequence list. The substitution can include, for example, making computer structural simulation analysis of the sequence, analyzing the potential existing post-translational modification (PTM) sites, especially in the Complementarity-Determining Regions (CDRs), including analyzing and substituting the sites of antibody aggregation, susceptible to asparagine deamidation (at sites like NG, NS, NH, etc.), susceptible to aspartic acid isomerization (at DG, DP sites), susceptible to N-glycosylation (at N-{P}S/T sites), and susceptible to oxidation.
“KD” refers to the dissociation constant derived from the ratio of Kd (the dissociation rate of specific binding molecule-target protein interaction) to Ka (the association rate of specific binding molecule-target protein interaction), or Kd/Ka, expressed in molar concentration (M).
The KD value can be determined using well-established methods in the art. A preferred method for determining the KD of a binding molecule is through the use of surface plasmon resonance, such as biosensor systems like the Biacore™ system (GE Healthcare Life Sciences). The term “treatment” or its equivalent expression, when used for, for example, cancer, refers to a procedure or process for reducing or eliminating the number of cancer cells in a patient or alleviating the symptoms of cancer. The “treatment” of cancer or another proliferative disorder does not necessarily mean that the cancer cells or other disorders will actually be eliminated, the number of cells or disorders will actually be reduced or the symptoms of cancers or other disorders will actually be alleviated. Generally, the method of treating cancer will be carried out even if it has only a low probability of success, but it is still considered to induce an overall beneficial course of action considering the patient's medical history and estimated survival expectation.
The term “pharmaceutically acceptable carrier” refers to any preparation or carrier medium capable of delivering an effective amount of the active substance of the present disclosure, without interfering with the biological activity of the active substance and causing no toxic side effects to the host or patient. Representative carriers include water, oil, vegetable and mineral, cream base, detergent base, ointment base, and the like. These bases may include suspending agents, thickeners, transdermal enhancers, etc. Their preparations are well known to those skilled in the arts of cosmetics or topical pharmaceutical.
The above preferred conditions can be combined arbitrarily to obtain preferred embodiments of the present disclosure without violating common knowledge in the art.
The reagents and raw materials used in the present disclosure are all commercially available.
The present disclosure's advantageous progressive effects include: the anti-DLL3 antibodies of the present disclosure possess excellent internalization activity, demonstrate superior binding activity with human DLL3 protein, and exhibit strong affinity at the protein level. The antibody-drug conjugates of the present disclosure exhibit excellent drug-forming properties, biological activity, and in vivo and in vitro anti-tumor activity, enabling the application of cytotoxic drugs in the treatment of patients with tumors characterized by neuroendocrine features, including SCLC.
The present disclosure will be further described below with reference to examples, but the present disclosure is not therefore limited to the scope of the examples. Experimental methods without specific conditions in the following examples are selected according to conventional methods and conditions, or according to the commercial specification.
In the present disclosure, monoclonal antibodies FDA027 and FDA031, which exhibit high affinity and specificity towards hDLL3, were selected. FDA027 comprises a light chain with an amino acid sequence as shown in SEQ ID NO: 1 and a heavy chain with an amino acid sequence as shown in SEQ ID NO: 2. FDA031 comprises a light chain with an amino acid sequence as shown in SEQ ID NO: 3 and a heavy chain with an amino acid sequence as shown in SEQ ID NO: 4.
The nucleotide sequences for the light and heavy chains of FDA027 were obtained through whole gene synthesis by Suzhou Genewiz. The sequences were individually cloned into the pV81 vector (as shown in
A human DLL3 expression plasmid, pCMV3-DLL3-t1 (purchased from Sino Biological Inc., HG20010-UT), was used to transform Escherichia coli competent cells, Trans1-T1 (purchased from TransGen Biotech, CD501), for the amplification of the transfection expression plasmid. The transformation process followed the instructions provided with the competent cells. Monoclones were picked from the transformation plates and expanded overnight in culture medium. The culture was then centrifuged at 6000 r/min for 20 minutes, and the bacterial solution was collected for plasmid extraction. A plasmid mid-prep extraction kit (purchased from Macherey-Nagel, product number: DP117) was used, according to the instruction manual to extract plasmid DNA.
Well-growing logarithmic phase HEK293 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were selected. Fresh culture medium, DMEM with 10% fetal bovine serum, was used to dilute the cells to a density of 5×105 cells/ml. 2 ml of the cell suspension was inoculated per well in a 6-well culture plate and cultured in an incubator (37° C., 5% CO2). The following day, the transfection reagent Lipofectamine 3000 (purchased from Thermo, L3000-008) was used to transfect the pCMV3-DLL3-t1 plasmid into HEK293 cells, following the instructions provided with the Lipofectamine 3000 transfection kit. 48 hours post-transfection, the cells were inoculated in 96-well culture plates at a density of 1 cell per well and cultured in fresh medium comprising 200 μg/ml G418 (purchased from Thermo, 10687010). The medium, comprising G418, was refreshed every 3 to 4 days to obtain stable monoclonal cells expressing hDLL3. Flow cytometry was used to screen HEK293 cells expressing hDLL3. The detection antibody, goat anti-hDLL3-PE (purchased from R&D, FAB4315P), was diluted with PBS to 10 μg/ml and 100 μl was added to 5×105 cells to be detected, followed by incubation at 4° C. for 1 hour. The cells were then resuspended in 1 ml of PBS comprising 2% fetal bovine serum, centrifuged at 1500 r/min for 5 minutes, and the supernatant was discarded, and the operation was repeated twice. The cells were resuspended in 1 ml of PBS and analyzed using a CytoFLEX flow cytometer (purchased from Beckman). Clones with a human DLL3 expression positivity rate greater than 85% were considered positive, and the HEK293-DLL3 stable cell line were established successfully.
HEK293 overexpression cell lines containing different hDLL3 domains were stably transfected using the same method as above, which including cell lines overexpressing hDLL3 without the N-terminal domain (sequence as shown in SEQ ID NO: 5), hDLL3 without the N-terminal and DSL domains (sequence as shown in SEQ ID NO: 6), hDLL3 without the N-terminal, DSL, and EGF1 domains (sequence as shown in SEQ ID NO: 7), and hDLL3 without the N-terminal, DSL, EGF1, and EGF2 domains (sequence as shown in SEQ ID NO: 8).
GKPIPNPLLGLDSTSGARCEPPAVGTACTRLCRPRSAPSRCGPGLRPCA
IAVIVVVVIAIVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA
GKPIPNPLLGLDSTSGAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLC
GGGGSGAGVIAVIVVVVIAIVAGIVVLVISRKKRMAKYEKAEIKEMGEM
HRELNA
GKPIPNPLLGLDSTSGGPGPCDGNPCANGGSCSETPRSFECTCPRGFYG
IAVIVVVVIAIVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA
GKPIPNPLLGLDSTSGSGVTCADGPCFNGGLCVGGADPDSAYICHCPPG
EMHRELNA
Six 6-week-old SPF grade female Balb/C mice (purchased from Shanghai Jihui Laboratory Animal Care Co., Ltd.) were selected and randomly divided into two groups, A and B. They were immunized with recombinant DLL3 protein (purchased from ACRO, DL3-H5255) at two-week intervals. Freund's complete adjuvant was used for the first immunization and Freund's incomplete adjuvant was used for subsequent immunizations. On days 35 and 49, blood samples were collected from the mice via orbital bleeding, and the immune serum titers of the mice were detected using conventional methods of ELISA and Flow Cytometry (FACS). ELISA detection method: DLL3 protein was diluted with PBS to a concentration of 1 μg/ml. 100 μl/well was added to a 96-well ELISA plate and incubated overnight at 4° C. The next day, the supernatant was discarded, and each well was blocked with 200 μl of 2% milk in PBS for 2 hours. After discarding the blocking solution, the wells were washed 3 times with 200 μl of PBST (0.1% Tween20). The serum to be detected was pre-diluted 100 times as the starting concentration, followed by a 3-fold gradient dilution across 11 concentrations, with 100 μl added to each well of the ELISA plate. After incubating at room temperature for 1 hour, the supernatant was discarded and the wells were washed 5 times with 200 μl of PBST (0.1% Tween20). Then, 100 μl of HRP-labeled goat anti-mouse secondary antibody (Jackson, 1:10000) was added and incubated at room temperature for 1 hour. After discarding the supernatant, the wells were washed 7 times with 200 μl of PBST (0.1% Tween20). 100 μl of TMB chromogen solution was added to each well for 10 minutes. The reaction was terminated with 2M HCl, and absorbance value at 450 nm was read with a Microplate Reader (purchased from Biotek, Elx808).
FACS detection method: 3×10{circumflex over ( )}5 HEK293-DLL3 stable cells or endogenously expressing cells SHP-77 (purchased from ATCC) were added to each well of a 96-well V-bottom plate (purchased from Axygen, wipp02280). The cells were centrifuged at 1500 r/min for 1 minute and the supernatant was discarded. The immunized serum was diluted with PBS at 1:50, followed by 4-fold gradient dilution across 8 concentrations, with 50 μL/well added to the plate. The cells were incubated on ice for 30 minutes, then resuspended in 150 μL of PBS, centrifuged at 1500 r/min for 1 minute, and the operation was repeated twice. APC-labeled goat anti-mouse IgG (Jackson, catalog number 115-605-164, diluted 1:800 in PBS) was added at 50 μL/well and incubated on ice for 30 minutes. The cells were then resuspended in 150 μL of PBS, centrifuged at 1500 r/min for 1 minute, and the operation was repeated twice. The samples were detected using a CytoFLEX flow cytometer (purchased from Beckman). After the detection, mice with the highest titer and consistent serum titer over two consecutive immunizations were selected for fusion. Three days before fusion, the mice were given a rush immunization via intraperitoneal injection with DLL3 protein.
Cell suspensions were prepared from the spleens of Balb/C mice that had received rush immunization. The spleen cells were fused with SP2/0 cells (purchased from the Cell Bank of the Chinese Academy of Sciences, TCM42), by electrofusion. After fusion, the hybridoma cells were inoculated at 2×104 cells per well in 96-well plates and selected using HAT medium. After 7 days, the supernatant from each well was detected using the ELISA method as described in Example 3-1. The ELISA-positive cells were further detected using FACS detection method to detect the 293 cell line overexpressing DLL3 and the SHP-77 endogenous cell line. The cell-binding positive clones were subjected to limited dilution and this process was repeated twice. The diluted clones were detected using the ELISA and FACS methods from Example 3-1 to confirm positive clones. The positive clone hybridomas detected after two limiting dilutions were expanded and used for antibody production and purification and antibody expression gene sequencing.
To produce milligram quantities of antibodies for functional characterization, selected hybridoma cells were inoculated at a density of 2.5×10{circumflex over ( )}5/mL in cell culture flasks comprising 250 mL of serum-free hybridoma medium (purchased from Yuanpei Biotechnology, H630KJ) and incubated at 37° C., shaking at 130 r/min. Once the cell viability dropped to around 30%, the supernatant was collected by centrifugation. Murine monoclonal antibodies were purified using Protein G media and dialyzed into PBS buffer, pH 7.2. The concentration and purity of the antibody were determined using a micro-volume spectrophotometer (purchased from Hangzhou Aosheng Instrument, Nano-300).
The SHP-77 cells were used to detect the internalization activity of the hybridoma antibodies obtained in Example 3-3 by using the FACS method described in Example 3-1. The detecting method is briefly described as follows: Six aliquots of 5×105 SHP-77 cells each were prepared in a 96-well V-bottom plate and centrifuged at 1500 r/min for 1 minute, after which the supernatant was discarded. 1 μg/ml of the saturated antibody to be detected was added to each well (200 μL per well) and incubated on ice for 30 minutes. Then, 150 μL of PBS was added to each well, followed by centrifugation at 1500 r/min for 1 minute, and the supernatant was discarded. This step was repeated four times. The cells were resuspended in 200 μl of PBS, and one aliquot was incubated at 37° C. for various durations: 5 hours, 3 hours, 1 hour, 0.5 hours, and 15 minutes. The remaining aliquot was kept on ice. After incubation, all samples were centrifuged at 4° C., 1500 r/min for 1 minute, and the supernatant was discarded. APC-labeled goat anti-mouse IgG (Jackson, catalog number 109-605-098, diluted 1:800 in PBS) was added to each well (50 μL per well) and incubated on ice for 30 minutes. The cells were then washed four times by resuspending in 150 μL of PBS, followed by centrifugation at 1500 r/min for 1 minute and discarding the supernatant. Finally, the cells were resuspended in 100 μL of PBS and analyzed using a CytoFLEX flow cytometer (purchased from Beckman). The fluorescence values for antibody internalizationare presented in Table 1.
From each clone, 2×105 hybridoma cells (originating from Example 3-2) were collected for RNA extraction. The cells were centrifuged at 300 g (the unit of rotational speed is used in this example, the same below) for 5 minutes and the supernatant was discarded. 250 μL of Trizol lysis buffer (purchased from TAKARA, T9108) was added to lyse the hybridoma cells. Then, 50 μL of chloroform was added, vortexed until the mixture turned milky, and allowed to stand at room temperature for 5 minutes. After centrifugation at 12000 g at 4° C. for 15 minutes, the supernatant was transferred to a new RNase-free 1.5 mL centrifuge tube. 125 μL of isopropanol was added, mixed well, and incubated at room temperature for 10 minutes. 12000 g, centrifuge for 10 minutes at 4° C. to see the precipitate, the supernatant was discarded, and 250 μL of 75% ethanol was added. The tube was gently inverted and centrifuged at 12000 g at 4° C. for 10 minutes, after which the supernatant was discarded, and the precipitate was air-dried at room temperature for 10 minutes. 20 μL of RNase-free water was then added to dissolve the precipitate for 30 minutes. After dissolution, it was mixed with a pipette and the concentration was determined. The RNA of the clones was reverse transcribed into cDNA using the PrimeScript™ RT Master Mix Kit (purchased from TAKARA, RR036A) according to the RNA concentration. Antibody light and heavy chain-specific primers were synthesized and used to obtain the antibody light and heavy chain sequences via PCR. The amplified fragments were gel recovered using the Universal DNA Purification Recovery Kit (purchased from TIANGEN Biotech, DP214-03), and then ligated with the pMD18T vector (purchased from TAKARA, 6011) at 4° C. for 30 minutes. 5 μL of the ligation product was added to 50 L of TG-1 competent cells (purchased from Lucigen), gently mixed, and subjected to heat shock at 42° C. for 90 seconds. The cells were then incubated on ice for 3 minutes, followed by the addition of 1 mL of 2 YT medium without antibiotics (purchased from Sangon Biotech, A507019-0250), and incubated at 37° C. on a shaker at 220 rpm for 1 hour. 100 μL of the culture was then spread on a 2 YT agar plate with Ampicillin resistance (purchased from Sangon Biotech, A600469-0005) and incubated at 37° C. for 14-16 hours. Monoclonies were picked for sequencing by Sangon Biotech. The sequencing results were analyzed using Vector NTI, Vbase2 software to obtain the amino acid sequences of the antibodies with good binding and internalization activities. The results are presented in Table 2.
For the hybridoma clones with good binding and internalization activities identified in Example 5, the heavy chain variable region sequences were synthesized and cloned into the pFUSEss-CHIg-hG1 vector (purchased from InvivoGen, catalog number pfusess-hchgl) comprising the amino acid sequence of the human antibody IgG1 heavy chain constant region by homologous recombination. This process resulted in the construction of chimeric antibody heavy chain expression vectors. Similarly, the cloned light chain variable region sequences were homologously recombined into the pFUSE2ss-CLIg-hk vector (purchased from InvivoGen, catalog number pfuse2ss-hclk) comprising the human antibody Kappa light chain constant region CL, obtaining the chimeric antibody light chain expression vectors. The amino acid sequence numbers corresponding to the full length of the light and heavy chains of the constructed chimeric antibodies are shown in Table 3-1. Well-growing logarithmic phase 293F cells were collected and inoculated into 250 mL cell culture flasks in 50 mL of culture medium. PEI was used to cotransfect 25 g each of the light and heavy chain expression plasmids. The supernatant on the 7th day post-transfection was collected, centrifuged, and filtered through a 0.45 M filter. The antibodies were purified using Protein A media and dialyzed into PBS buffer, pH 7.2. The concentration of the antibodies was determined using a micro-volume spectrophotometer (purchased from Hangzhou Aosheng Instrument, Nano-300). The binding activity of the obtained chimeric antibodies to human DLL3 protein was detected using the ELISA method (results shown in
The chimeric antibodies obtained in Example 6 were labeled with Biotin, and their competition binding activities were detected through the ELISA method. The detection method is briefly described as follows: The concentration of DLL3 protein was diluted with PBS to 1 μg/ml and coated onto a 384-well ELISA plate (purchased from Corning, 3700) overnight. The next day, the supernatant in the plate wells was discarded, and the wells were blocked with 80 μl of 3% milk (dissolved in PBS) for 2 hours. The wells were then washed three times with 80 μl of PBST (0.1% Tween20). Afterwards, the unlabeled chimeric antibodies were pre-diluted to 100 μg/ml and further diluted in a 3-fold gradient across 11 concentration points, with 25 μl added to each well of the previously blocked ELISA plate, incubated at room temperature for 1 hour, after which the supernatant was discarded, and the wells were washed five times with 80 μl of PBST (0.1% Tween20). Then, 25 μl of the Biotin-labeled chimeric antibodies were added and incubated at room temperature for 1 hour, followed by another round of washing with 80 μl of PBST (0.1% Tween20) five times. Finally, HRP-labeled streptavidin secondary antibody (1:5000, purchased from Kingfisher Biotech, M00091) was added and incubated at room temperature for 1 hour, after which the supernatant was discarded, and the wells were washed seven times with 80 μl of PBST (0.1% Tween20). Then, 25 μl of TMB chromogen solution was added for 10 minutes, and the reaction was terminated with 2M HCl. The absorbance value at 450 nm was read with a Microplate reader (purchased from Biotek, Elx808). To determine the specific binding regions of the antibodies, HEK293 cell lines overexpressing different hDLL3 structural domains (corresponding sequences: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8) were analyzed by FACS to determine the binding ability of the chimeric antibodies to these four cell types, identifying the antibody binding regions. In brief: 5×105 cells overexpressing DSL, EGF1, EGF2, and EGF3 were placed in 96-well V-bottom plates. The cells were centrifuged at 1500 rpm for 1 minute and the supernatant was discarded. 1 μg/ml of the saturated antibody to be detected was added to each well (200 μL per well) and incubated on ice for 30 minutes. Then 150 μL of PBS was added to each well, centrifuged at 1500 r/min for 1 min, the supernatant was discarded, and the plate washing was repeated 4 times. APC-labeled goat anti-human IgG (Jackson, diluted 1:800 in PBS) was added to each well (50 μL per well) and incubated on ice for 30 minutes. After washing with PBS four times, 100 μL PBS was added to each well to resuspend the cells, which were then analyzed using CytoFLEX (purchased from Beckman). The results showed that antibodies ch1A5, ch33F11, and ch32E8 bind to the EGF2 domain of DLL3; FDA031, ch13C2, and ch15G2 bind to the N-terminal of DLL3; FDA027 binds to the DSL domain of DLL3; and the remaining antibodies bind to the EGF3-EGF6 domains of DLL3. Thus, the binding epitope of ch15G2 is identified as the N-terminal domain; the binding epitope of ch32E8, ch1A5, and ch33F11 is the EGF2 domain; and the binding epitopes of ch36H10, ch6A2, ch19C7, ch26D9, ch23G2, ch10B1, and ch4H7 are the EGF3-EGF6 domains.
The chimeric antibodies obtained in Example 6 were evaluated for their binding abilities to human DLL1 (purchased from Acro, DL1-H52H8) and DLL4 (purchased from Acro, DL4-H5227) proteins using the ELISA method. The ELISA detection method involved diluting DLL1 and DLL4 proteins with PBS to a final concentration of 1 μg/ml and coating them onto 384-well ELISA plates (purchased from Corning, 3700) at 4° C. overnight. The next day, the supernatant in the plate wells was discarded, and the wells were blocked with 80 μl of 3% milk (dissolved in PBS) for 2 hours, followed by washing three times with 80 μl of PBST (0.1% Tween20). The chimeric antibodies to be detected, FDA027, and FDA031 were then diluted to 20 μg/ml respectively, and further diluted in a 3-fold gradient across 11 concentration points, with 25 μl added to each well of the previously blocked ELISA plate, and incubated at room temperature for 1 hour. After washing with 80 μl of PBST (0.1% Tween20) for five times, 25 μl of HRP-labeled goat anti-human secondary antibody (Jackson, 1:10000) was added to each well and incubated at room temperature for 1 hour, discard the supernatant, and the wells were then washed seven times with 80 μl of PBST (0.1% Tween20). After adding 25 μl of TMB chromogen solution for 10 minutes, the reaction was terminated with 2M HCl, and the absorbance value at 450 nm was measured with a Microplate reader (purchased from Biotek, Elx808). The results (shown in
To assess the affinity of antibodies for human DLL3 protein, this study employed the BLI method to detect the binding kinetics curves of immobilized antibodies with free DLL3. The method was conducted according to the instrument's user manual (supplier: Fortebio, equipment model: Octet 96e). Briefly, AMC sensors were equilibrated for 60 s with Loading Buffer/Sample dilution buffer (1×PBS, pH7.4, with 0.1% BSA and 0.02% Tween-20) to obtain Baseline 1. The antibodies to be detected were diluted with Loading Buffer to a concentration of 10 ug/ml and bound to the equilibrated sensors, after which the sensors were re-equilibrated with Loading Buffer to obtain Baseline 2. The antibody-loaded sensors were then placed in human DLL3, His tag (supplier: acrobiosystems, catalog number: DL3-H52H4) diluted with Sample Dilution Buffer to concentrations ranging from 100 to 3.13 nM for 90 s to obtain the antibody-protein binding curve. Then, the sensors combined with antigen were placed in the Sample Dilution Buffer again and dissociated for 180 s, and the dissociation curve was obtained. The k-on and k-off values of the antibody-protein binding were calculated based on the binding and dissociation curves, and the KD values were calculated, with the results shown in Table 4. The results indicated that these chimeric antibodies exhibited strong affinity at the protein level.
The linker-drug conjugates used in the present disclosure, LE00 and LE01-LE24, are outlined in Table 5. LE00 (GGFG-Dxd) was synthesized following the method reported in WO2015146132A, and LE01-LE24 were synthesized following the methods reported in WO2020259258A1.
Different anti-DLL3 antibodies obtained from Examples 1 and 6 were exchanged into a 50 mM PB/1.0 mM EDTA buffer (pH 7.0) using G25 desalting columns. Then, 12 equivalents of TECP were added and the mixture was stirred at 37° C. for 2 hours to fully open the inter-chain disulfide bonds of the antibodies. The pH of the reduced antibody solution was subsequently adjusted to 6.0 using phosphoric acid, and the water bath temperature was lowered to 25° C. to prepare for the conjugation reaction. The linker-drug conjugates prepared as per Example 10 were dissolved in DMSO, and 12 equivalents of the linker-drug conjugate were added dropwise to the reduced anti-DLL3 antibody solution respectively. Each sample was prepared according to the maximum Drug-to-Antibody Ratio (DAR)((i.e. excessive coupling), observing the formation of any precipitates during the conjugation reactions. DMSO was added to reach a final concentration of 10% (v/v), and the mixture was stirred at 25° C. for 0.5 hours. After the reaction, the samples were filtered through a 0.22 μm membrane. Unconjugated small molecules were removed by purification using a tangential flow ultrafiltration system, with the buffer being 50 mM PB/1.0 mM EDTA solution (pH 6.0). After the purification, a final concentration of 6% sucrose was added, and the samples were stored at −20° C. The UV method was used to measure absorbance values at 280 nm and 370 nm, from which DAR values were calculated. The results are shown in Table 6. The results indicate that the antibodies ch1A5, ch33F11, ch32E8, ch13C2, FDA027, FDA031, ch15G2, and ch4H7 were successfully conjugated with LE14. ch33F11 was successfully conjugated with LE01-LE11, LE23, and LE24; ch1A5 was successfully conjugated with LE00, LE12, LE13, LE15, LE16, LE17, LE18, LE19, LE20, LE21 and LE22. The DAR values and free Dxd contents of the resulting ADCs met the expected requirements. However, antibodies ch19C7, ch23G2, ch6A2, and ch26D9 showed noticeable flocculent precipitation during centrifugal concentration when conjugated with LE14, resulting in a lower yield and indicating that their drug conjugation was not feasible. The ADC samples from ch36H10 and ch10B1 had DAR values of 1.89 and 1.90, respectively, both had high impurity contents. Particularly, ch36H10 had a high residual of small molecules (49.35%), indicating a failed conjugation.
HEK293 cells stably transfected with DLL3 with high expression were selected as cell lines for in vitro activity detection, and the dose-response of different antibody drug conjugates on cell killing was observed. 2000 cells per well were inoculated in a 96-well cell culture plate and cultured for 20-24 hours. The antibody drug conjugates prepared according to the method described in Example 11 were diluted to a series of 9 concentration gradient test solutions: 80, 20, 5, 1.25, 0.3125, 0.0781, 0.0195, 0.00488, and 0.000488 μg/ml. 100 μl per well of these diluted test solutions were added to the wells comprising the inoculated cells. The plates were then incubated in a 37° C., 5% CO2 incubator for 144 hours. Afterwards, CellTiter-Glo® Luminescent Cell Viability Assay Reagent (50 μl/well) was added, and the plates were shaken at 500 rpm at room temperature for 10 minutes to ensure mixing. The data was read using a SpectraMaxL Microplate reader (OD570 nm, with a 2-second interval between readings). The viability of the untreated control cells was considered as 100% for data processing, and the IC50 values were calculated. The results are presented in
1 and 2 indicate a mean value.
The results from Table 7 indicate that, with the exception of ch19C7-LE14 and ch13C2-LE14, which showed relatively weaker in vitro cytotoxic activity, all ADCs disclosed in the present invention demonstrated significant in vitro cell-killing effects on DLL3-positive cells. Among these, FDA027-LE14 exhibited the strongest in vitro cytotoxic activity, and also had the most optimal IC50 value in terms of in vitro activity. Furthermore, the IC90 values of the various ADCs disclosed in the present disclosure did not show significant differences when compared to FDA027-LE14.
Human small cell lung cancer cells (NCI-H82), suspended in 200 μl Matrigel, were subcutaneously injected into the right dorsal side of female Balb/c nude mice (6-8 weeks old, purchased from Shanghai Lingchang Biotechnology Co., Ltd.). When the average tumor volume reached approximately 140 mm3, the mice were randomly divided into 15 groups based on tumor size and body weight, with 8 animals in each group of 1-13, which are: vehicle control group; ch1A5-LE14, ch33F11-LE14, ch23G2LE14, ch32E8-LE14, FDA031-LE14, and FDA027-LE14 conjugates at two dosage levels (2.5 mg/kg and 5.0 mg/kg), administered intravenously once a week for a total of three doses. Groups 14-15, each with 6 animals, served as control groups receiving Lurbinectedin (0.18 mg/kg) and Topotecan Hydrochloride (1.8 mg/kg), respectively. Group Lurbinectedin was administered intravenously once a week for a total of three doses, while Group Topotecan Hydrochloride was administered intraperitoneally twice a week (continuously on the 1st and 2nd days) for a total of three doses. The body weight and tumor volume of the experimental animals were measured thrice weekly, and their survival status was monitored throughout the experiment. The results, as depicted in
Female Balb/c nude mice, aged 6-8 weeks, were subcutaneously injected on the right dorsal side with 1×107 human small cell lung cancer cells (NCI-H209) suspended in 200 μL of Matrigel. When the average tumor volume reached approximately 150 mm3, the mice were randomly divided into seven groups based on tumor size and body weight, with six animals per group. The groups included a vehicle control group, and three dosage groups for both of the conjugates ch1A5-LE14 and FDA027-LE14, specifically at 1.5 mg/kg, 2.5 mg/kg, and 5.0 mg/kg, administered as a single dose. The body weight and tumor volume of the experimental animals were measured thrice weekly, and their survival status was monitored throughout the experiment. The results are presented in Table 9. The vehicle control group (Group 1) had an average tumor volume of 2006 mm3 at the end of treatment. The 1.5 mg/kg FDA027-LE14 treatment group (Group 5), the 2.5 mg/kg FDA027-LE14 group (Group 6), and the 5 mg/kg FDA027-LE14 group (Group 7) all exhibited an average tumor volume of 0 mm3 on Day 21 post-treatment. The 1.5 mg/kg ch1A5-LE14 treatment group (Group 2), the 2.5 mg/kg ch1A5-LE14 group (Group 3), and the 5 mg/kg ch1A5-LE14 group (Group 4) showed an average tumor volume of 0 mm3 on Day 21 post-treatment. The experimental results indicated that both FDA027-LE14 and ch1A5-LE14 demonstrated significant in vivo antitumor activity, and all experimental mice survived without weight loss, indicating good safety profiles of the test drugs.
Mice of the KM strain were selected for toxicity evaluation. The mice were randomly divided into four groups, each consisting of ten animals, with an equal number of males and females. Each group received a single intravenous injection of 600 μL of the ch1A5-LE14 conjugate at doses of 200 mg/kg, 400 mg/kg, 500 mg/kg, and 640 mg/kg. Toxicity responses to the ch1A5-LE14 conjugate were observed over a period of 14 days post-administration. At 200 mg/kg, one animal exhibited a temporary decrease in body weight on Day 3, which then recovered and increased; other animals showed a growth trend throughout the experiment. Clinical observations showed no abnormalities, except for the 200 mg/kg group, and the mice of other groups displayed transient fur fluffing on the day of administration and recovered quickly. By the end of the 14-day observation period, all animals survived without dying and death, and the results showed that the conjugates of the present invention was well tolerated in mice, and the maximum tolerated dose was up to 640 mg/kg. In another study evaluating the toxicity of the ch1A5-LE14 conjugate in KM mice, the intraperitoneal injection doses were 410 mg/kg, 512 mg/kg, 640 mg/kg, 800 mg/kg, and 1000 mg/kg, respectively. Symptoms observed included fur fluffing, arched backs, reduced activity, huddling, diarrhea, soft stools, and perianal filth. In the 1000 mg/kg group, five out often mice died, and in the 800 mg/kg group, one mouse underwent euthanasia after experiencing a body weight decrease exceeding 30% on three consecutive occasions during the 14-day observation period, in accordance with animal welfare guidelines. No deaths were observed in the other groups, further demonstrating the excellent tolerance of the conjugate in mice.
SD rats were selected for intravenous injection of the ch1A5-LE14 conjugate to assess tolerance in rats. The rats were randomly divided into three groups, each consisting of eight animals, with an equal number of males and females. One group received 100 mg/kg once a week for three weeks, another group received a single dose of 300 mg/kg, and a blank control group was established. The volume of administration was adjusted to 10 μL/g of rat body weight, and observations were made for 12 days following the last administration. At the end of the 12-day observation period post-last administration, no dying or death incidents occurred in any group. All animals in the high-dose 300 mg/kg group exhibited fur fluffing post-administration, while no abnormalities were observed in the 100 mg/kg group. The body weight in the 300 mg/kg group decreased post-administration but showed a recovery trend from Day 7 onwards. All animals were euthanized for autopsy at the end of the observation period, and no abnormal findings were observed in the organs. The results of the toxicity study in rats further demonstrate that the conjugate of the present disclosure exhibits a very good tolerance level in rats.
Although specific embodiments of the present disclosure have been described, it should be understood by those skilled in the art that these are provided for illustrative purposes only. Various changes or modifications can be made without departing from the principles and essence of the present disclosure. Therefore, the scope of the present disclosure is defined by the attached claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/109517 | Jul 2021 | WO | international |
| 202210239591.2 | Mar 2022 | CN | national |
The present application claims priority to PCT patent application PCT/CN2021/109517 filed on Jul. 30, 2021, and Chinese patent application CN2022102395912 filed on Mar. 11, 2022. The contents of the above patent applications are incorporated herein by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/109050 | 7/29/2022 | WO |
