Humanized anti-CD22 recombinant immunotoxin and its uses

Abstract

Humanized anti-CD22 recombinant immunotoxins are provided that have reduced immunogenicity, maintain the CD22 binding affinity and corresponding biological activity prior to humanization, and are used to prepare drugs for treating CD22-associated B-cell malignancies. TIFF2023544457000041.tif22164

Description

Technical Field

[0001] This application claims the benefit of priority of Chinese Patent Application No. CN202011048723.0, filed on September 29, 2020, the entire disclosure of which is incorporated herein by reference.

[0002] Technical Field The present invention relates to the field of biomedicine, and specifically to a recombinant immunotoxin of a humanized anti-CD22 antibody fragment fused with truncated Pseudomonas exotoxin A and its uses.

Background Art

[0003] Background of the Invention CD22 (Siglec-2) is a sialic acid-binding immunoglobulin-like lectin (Siglec) receptor that specifically binds to sialic acid (Sia)-containing glycans and promotes cell adhesion and / or cell signaling. [1] CD22 expression is restricted to B cells and plays an important role in establishing the baseline level of B cell inhibition. Thus, CD22 is an important determinant of homeostasis in humoral immunity. CD22 is expressed on tumor cells at varying rates in B cell malignancies such as B lymphoblastic leukemia / lymphoma and mature B cell leukemia / lymphoma. [2] CD22 expression can be particularly strong in hairy cell leukemia (HCL) and prolymphocytic leukemia.

[0004] Hairy cell leukemia (HCL), first described by Bouroncle et al. in 1958, is a chronic malignancy of mature neoplastic B cells with characteristic serrated cytoplasmic margins. [3、4] HCL accounts for 2% of all leukemias in the United States (500 - 800 new cases per year in the United States [5] ) and is characterized by pancytopenia and splenomegaly. Purine analogues (cladribine or pentostatin) are the standard treatment for initial treatment of HCL and are associated with durable remissions lasting several years; however, many patients relapse and require additional treatment. [6]Subsequent treatments generally follow a modified schedule of purine analogs, but the efficacy of the treatment decreases, the patient has a shorter remission, and ultimately becomes resistant to the treatment. [7] Furthermore, purine analogs are associated with neurotoxicity [8] and are highly immunosuppressive, which can increase the risk of opportunistic infections. [4] 。

[0005] Immunotoxins have been proven to be effective therapeutic agents for hairy cell leukemia. Immunotoxins are antibody-conjugated therapeutic agents that use a potent cytotoxic payload, such as a bacterial toxin [9] , a plant-derived toxin

[10] , and a synthetic chemical

[11] to target and kill cancer cells. The first generation of anti-CD22 immunotoxins was developed at the National Cancer Institute in the late 1990s and reported under the name BL22 (RFB4(dsFv)-PE38 or CAT-3888). A mouse anti-CD22 antibody fused with a 38 kDa fragment of Pseudomonas exotoxin A (PE38) was utilized. CAT-3888 entered a Phase I trial and reported remission of leukemia in 2001. [12-16] However, CAT-3888 has been replaced by an improved immunotoxin, moxetumomab pasudotox (HA22 or CAT-8015), which includes modifications to PE38 and the anti-CD22 antibody fragment. In HA22, three amino acids within the antibody fragment were changed compared to CAT-3888 to increase the binding affinity for the target molecule.

[0006] HA22 is composed of the Fv fragment of a mouse anti-CD22 monoclonal antibody fused to PE38. Mechanistically, the Fv portion of HA22 binds to CD22, a cell surface receptor expressed on diverse malignant B cells, thereby delivering the toxin moiety PE38 directly to tumor cells. After internalization, PE38 catalyzes the ADP-ribosylation of the diphthamide residue of elongation factor 2 (EF-2), resulting in a rapid decrease in the level of the anti-apoptotic protein, myeloid cell leukemia sequence 1 (Mcl-1), and causing apoptotic cell death. [17、18] HA22 was approved by the US FDA in 2018 for the treatment of adults with relapsed or refractory hairy cell leukemia who have received at least two prior systemic therapies, such as treatment with purine nucleoside analogs.

[0007] Initial immunotoxin designs lacked a sufficient therapeutic window to warrant further clinical development, and in particular, inherent immunogenicity and the rapid development of anti-drug antibodies were important factors.

[19] The targeting moiety contained in HA22, namely the Fv portion of the mouse anti-CD22 monoclonal antibody, is of murine origin and thus has some immunogenicity in humans

[20] and thereby causes the production of unwanted neutralizing anti-drug antibodies (ADA), which negatively affect the pharmacokinetic profile, for example, affecting the activity of the drug at a given dosage, accelerating the clearance of the drug in vivo, and limiting the duration and effectiveness of repeated dosing. SUMMARY OF THE INVENTION

[0008] To solve the above technical problems, an object of the present invention is to provide a novel recombinant immunotoxin based on Pseudomonas exotoxin A. In the recombinant immunotoxin, a humanized anti-CD22 monoclonal antibody fragment having reduced immunogenicity compared to the original mouse-derived anti-CD22 monoclonal antibody fragment and retaining the affinity for the target CD22 is fused with Pseudomonas exotoxin A; at the same time, the formed novel recombinant immunotoxin will have a strong immunotoxin efficacy, such as a growth inhibitory effect and an apoptosis effect, on CD22-expressing tumor cells.

[0009] A further object of the present invention is to provide the use of the novel recombinant immunotoxin.

[0010] The technical solution of the present invention is as follows.

[0011] In one aspect, the present disclosure provides a humanized anti-CD22 recombinant immunotoxin, which is a polypeptide molecule comprising the following two polypeptide chains: (1) A first polypeptide chain comprising the variable light chain region (V L ) of an anti-CD22 antibody, wherein the variable light chain region (V L ) comprises the amino acid sequence shown in SEQ ID NO.16 or SEQ ID NO.18, or an amino acid sequence homolog thereof having at least 75% sequence identity with the amino acid sequence shown in SEQ ID NO.16 or SEQ ID NO.18; (2) A second polypeptide chain comprising the variable heavy chain region (V H ) of an anti-CD22 antibody and a cytotoxic agent directly or indirectly linked to the variable heavy chain region (V H ), wherein the variable heavy chain region (V H ) comprises the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4, or an amino acid sequence homolog thereof having at least 75% identity with the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4.

[0012] As used herein, "amino acid sequence homolog" refers to an amino acid sequence that is derived from the corresponding amino acid sequence but has one or more amino acid substitutions, additions, or deletions compared thereto.

[0013] In the first polypeptide chain, preferably, the amino acid sequence homolog has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO.16 or SEQ ID NO.18. More preferably, the amino acid sequence homolog includes the amino acid sequences shown in SEQ ID NO.31, SEQ ID NO.32, and SEQ ID NO.33, and has the same amino acid residues as SEQ ID NO.16 or SEQ ID NO.18 at positions 3, 5, 36, 37, 47, 48, 49, 50, 65, 67, 69, 70, and 72 corresponding to the amino acid sequence shown in SEQ ID NO.14 (the amino acid positions are numbered according to the amino acid sequence shown in SEQ ID NO.14), but is not the amino acid sequence shown in SEQ ID NO.14.

[0014] In the second polypeptide chain, preferably, the amino acid sequence homolog has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4. More preferably, the amino acid sequence homolog includes the amino acid sequences shown in SEQ ID NO.28, SEQ ID NO.29, and SEQ ID NO.30, and has the same amino acid residues as SEQ ID NO.3 or SEQ ID NO.4 at positions 3, 48, 49, 50, 69, 71, 73, 75, and 80 corresponding to the amino acid sequence shown in SEQ ID NO.2 (the amino acid positions are numbered according to the amino acid sequence shown in SEQ ID NO.2), but is not the amino acid sequence shown in SEQ ID NO.2.

[0015] Preferably, in the humanized anti-CD22 recombinant immunotoxin according to the present invention, the light chain variable region (V L ) of the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO.16 or an amino acid sequence homolog thereof; the heavy chain variable region (V H ) of the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO.3 or an amino acid sequence homolog thereof.

[0016] Preferably, in the humanized anti-CD22 recombinant immunotoxin according to the present invention, the light chain variable region (V L ) of the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO.18 or an amino acid sequence homolog thereof; the heavy chain variable region (V H ) of the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO.3 or an amino acid sequence homolog thereof.

[0017] Preferably, in the humanized anti-CD22 recombinant immunotoxin according to the present invention, the light chain variable region (V L) comprises the amino acid sequence shown in SEQ ID NO.16 or an amino acid sequence homolog thereof; the heavy chain variable region (V of the second polypeptide chain H ) comprises the amino acid sequence shown in SEQ ID NO.4 or an amino acid sequence homolog thereof.

[0018] Preferably, in the humanized anti-CD22 recombinant immunotoxin according to the present invention, the light chain variable region (V of the first polypeptide chain L ) and the heavy chain variable region (V of the second polypeptide chain H ) are covalently linked, for example, via a disulfide bond.

[0019] Preferably, in the humanized anti-CD22 recombinant immunotoxin according to the present invention, the cytotoxin of the second polypeptide chain is Pseudomonas exotoxin A, or a mutant or fragment of Pseudomonas exotoxin A in which cytotoxicity is retained.

[0020] Naturally occurring Pseudomonas exotoxin A (PE) is a bacterial toxin secreted by Pseudomonas aeruginosa. It is a monomeric protein with a molecular weight of 66 kD. In order to reduce or eliminate the non-specific binding of the toxin while maintaining cytotoxicity, the PE molecule can be modified or a part of its sequence can be removed. For example, a mutant of Pseudomonas exotoxin A is a modified Pseudomonas exotoxin A that retains cytotoxicity, and the modification can be a conservative modification. For example, the mutant has at least 75%, preferably at least 80%, more preferably at least 90%, 91%, 92%, 93%, 94%, 95% amino acid sequence identity with unmodified native PE. Also, for example, a fragment of Pseudomonas exotoxin A is a shortened form of Pseudomonas exotoxin A that retains cytotoxicity. Preferably, the mutant or fragment of Pseudomonas exotoxin A according to the present invention is, for example, PE40, PE38, PE35, PE24, mPE24, or T19, T20, M11 [21、22、23、24、25] . According to one specific embodiment of the present invention, the fragment is PE38 having the amino acid sequence shown in SEQ ID NO.1.

[0021] Preferably, in the second polypeptide chain, the cytotoxin is fused to the C-terminus of the variable region (V H ) of the heavy chain of the anti-CD22 antibody. For example, the N-terminus of the cytotoxin is fused to the C-terminus of the variable region (V H ) of the heavy chain, either directly or via a linker. The linker can form a covalent bond with the variable region (V H ) of the heavy chain and the cytotoxin, respectively, without affecting the function of each functional component of the humanized anti-CD22 recombinant immunotoxin. Suitable linkers, such as linear or branched carbon linkers, heterocyclic carbon linkers, and peptide linkers, are known in the art. In the humanized anti-CD22 recombinant immunotoxin according to the present invention, preferably, no linker is used or a peptide linker, i.e., a linking peptide, such as a flexible linking peptide containing one or more (GGGGS) amino acid sequences, is used.

[0022] In the humanized anti-CD22 recombinant immunotoxin according to the present invention, the first polypeptide chain and the second polypeptide chain may form the form of a Fab, i.e., the first polypeptide chain further comprises a κ light chain constant region fused to the C-terminus of the variable region (V L ) of the light chain, preferably a human κ light chain constant region; or the second polypeptide chain further comprises a heavy chain constant region CH1 fused to the C-terminus of the variable region (V H ) of the heavy chain, preferably a human heavy chain constant region CH1.

[0023] According to one specific embodiment of the present invention, in the humanized anti-CD22 recombinant immunotoxin according to the present invention, the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO.16 or an amino acid sequence homolog thereof, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO.9 or an amino acid sequence homolog thereof.

[0024] Alternatively, the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 18 or an amino acid sequence homolog thereof, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 9 or an amino acid sequence homolog thereof.

[0025] Alternatively, the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 16 or an amino acid sequence homolog thereof, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 10 or an amino acid sequence homolog thereof.

[0026] In another aspect, the present disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding the first polypeptide chain of the humanized anti-CD22 recombinant immunotoxin according to the present invention and / or a nucleotide sequence encoding the second polypeptide chain of the humanized anti-CD22 recombinant immunotoxin according to the present invention. The nucleic acid molecule according to the present invention may be a single nucleotide sequence encoding the first polypeptide chain or the second polypeptide chain; or, the nucleic acid molecule according to the present invention may be a single nucleotide sequence encoding both the first polypeptide chain and the second polypeptide chain. Alternatively, the nucleic acid molecule according to the present invention may be a combination of two nucleotide sequences encoding the first polypeptide chain and the second polypeptide chain, respectively.

[0027] In yet another aspect, the present disclosure provides a vector comprising the nucleic acid molecule according to the present invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, etc. According to one specific embodiment of the present invention, the vector is a prokaryotic expression vector containing the nucleic acid molecule, for example, an expression plasmid.

[0028] A map of a plasmid according to one specific embodiment of the present invention containing nucleotide sequences encoding the first polypeptide chain and the second polypeptide chain is shown in FIG. 3.

[0029] In yet another aspect, the present disclosure provides a host cell. The host cell has been transformed or transfected with a nucleic acid molecule and / or vector according to the present invention and thus contains the nucleic acid molecule and / or vector of the present invention for storage or expression. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, or insect, fungal, plant, or animal cell. According to one specific embodiment of the present invention, the host cell is a prokaryotic cell, such as an Escherichia coli (E. coli) cell.

[0030] According to the disclosure of the present invention, the humanized anti-CD22 recombinant immunotoxin, nucleic acid molecule, vector, and / or host cell according to the present invention may be obtained by utilizing any conventional technique known in the art. For example, by using the nucleic acid molecule or vector according to the present invention, the first polypeptide chain and the second polypeptide chain can be expressed in a host cell, and then, by recovery of the chains and in vitro refolding, the light chain variable region (V L ) of the first polypeptide chain is linked via a disulfide bond to the heavy chain variable region (V H ) of the second polypeptide chain to form a heterodimer.

[0031] The humanized anti-CD22 recombinant immunotoxin, nucleic acid molecule, vector, and / or host cell according to the present invention may be contained in a composition, such as a pharmaceutical composition, more specifically a pharmaceutical preparation, for use for various purposes according to actual needs.

[0032] Accordingly, in a further aspect, the present disclosure also provides a composition comprising a humanized anti-CD22 recombinant immunotoxin, nucleic acid molecule, vector, and / or host cell according to the present invention. Preferably, the composition is a pharmaceutical composition optionally containing a pharmaceutically acceptable excipient.

[0033] The present disclosure further provides the following related uses of the above-described subject matter based on a humanized anti-CD22 recombinant immunotoxin that can bind to a target CD22, specifically human CD22, which is a B cell surface marker.

[0034] Specifically, in one aspect, the present disclosure provides the use of a humanized anti-CD22 recombinant immunotoxin, nucleic acid molecule, vector, host cell, or composition in the manufacture of a medicament for the treatment of CD22-related B-cell malignancies.

[0035] Preferably, the CD22-related B-cell malignancies are B-cell malignancies characterized by high CD22 expression, such as lymphomas or leukemias having high CD22 expression. Preferably, the lymphoma is non-Hodgkin lymphoma, small lymphocytic lymphoma, or mantle cell lymphoma; and the leukemia is chronic lymphocytic leukemia, hairy cell leukemia, or acute lymphocytic leukemia.

[0036] In another aspect, the present disclosure provides a method for treating CD22-related B-cell malignancies, comprising the step of administering a humanized anti-CD22 recombinant immunotoxin, nucleic acid molecule, vector, and / or host cell to a subject in need thereof. The subject is a mammal, such as a human or non-human primate, and a companion animal, livestock, or laboratory mammal, such as a dog, cat, cow, pig, sheep, horse, mouse, and rabbit. Preferably, the subject is a human.

[0037] The humanized V H and V L may form another dsFv antibody. Thus, in yet another aspect, the present disclosure provides a humanized anti-CD22 antibody or antibody fragment comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein the heavy chain variable region (V H ) comprises the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4, or an amino acid sequence homolog thereof having at least 75% identity with the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4, and the light chain variable region (V L) comprises an amino acid sequence shown in SEQ ID NO.16 or SEQ ID NO.18, or an amino acid sequence homolog thereof having at least 75% identity with the amino acid sequence shown in SEQ ID NO.16 or SEQ ID NO.18, and provides a humanized anti-CD22 antibody or antibody fragment.

[0038] In the light chain variable region (V L ), preferably, the amino acid sequence homolog has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO.16 or SEQ ID NO.18. More preferably, the amino acid sequence homolog comprises the amino acid sequences shown in SEQ ID NO.31, SEQ ID NO.32, and SEQ ID NO.33, and has the same amino acid residues as SEQ ID NO.16 or SEQ ID NO.18 at positions 3, 5, 36, 37, 47, 48, 49, 50, 65, 67, 69, 70, and 72 corresponding to the amino acid sequence shown in SEQ ID NO.14 (the amino acid positions are numbered according to the amino acid sequence shown in SEQ ID NO.14), but is not the amino acid sequence shown in SEQ ID NO.14.

[0039] In the heavy chain variable region (V H) Preferably, in the humanized anti-CD22 antibody or antibody fragment according to the present invention, the amino acid sequence homolog has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4. More preferably, the amino acid sequence homolog includes the amino acid sequences shown in SEQ ID NO.28, SEQ ID NO.29, and SEQ ID NO.30, and has the same amino acid residues as SEQ ID NO.3 or SEQ ID NO.4 at positions 3, 48, 49, 50, 69, 71, 73, 75, and 80 corresponding to the amino acid sequence shown in SEQ ID NO.2 (the amino acid positions are numbered according to the amino acid sequence shown in SEQ ID NO.2), but is not the amino acid sequence shown in SEQ ID NO.2.

[0040] Preferably, in the humanized anti-CD22 antibody or antibody fragment according to the present invention, the light chain variable region (V L ) includes the amino acid sequence shown in SEQ ID NO.16 or an amino acid sequence homolog thereof; the heavy chain variable region (V H ) includes the amino acid sequence shown in SEQ ID NO.3 or an amino acid sequence homolog thereof.

[0041] Preferably, in the humanized anti-CD22 antibody or antibody fragment according to the present invention, the light chain variable region (V L ) includes the amino acid sequence shown in SEQ ID NO.18 or an amino acid sequence homolog thereof; the heavy chain variable region (V H ) includes the amino acid sequence shown in SEQ ID NO.3 or an amino acid sequence homolog thereof.

[0042] Preferably, in the humanized anti-CD22 antibody or antibody fragment according to the present invention, the light chain variable region (V L ) includes the amino acid sequence shown in SEQ ID NO.16 or an amino acid sequence homolog thereof; the heavy chain variable region (V H) comprises the amino acid sequence shown in SEQ ID NO.4 or an amino acid sequence homolog thereof.

[0043] Preferably, the humanized anti-CD22 antibody according to the present invention is a monoclonal antibody further comprising a human heavy chain constant region (CH) and a light chain constant region (CL); preferably, the monoclonal antibody comprises a heavy chain (HC) and a light chain (LC).

[0044] Depending on the heavy chain constant region sequence of the monoclonal antibody, the antibody can be of the IgA class, IgD class, IgE class, IgG class, or IgM class, and further can be of a subclass (isotype) such as IgG1, IgG2, IgG3, or IgG4. Depending on the light chain constant region sequence of the monoclonal antibody, the antibody can be of the κ type or λ type.

[0045] Preferably, the antibody fragment according to the present invention is a monoclonal antibody fragment that recognizes CD22 or retains the ability to bind to CD22, preferably Fab, Fab', F(ab')2, scFv, dsFv, or any other antibody fragment. Depending on the context, "antibody fragment" can be used interchangeably with "antibody" in the present disclosure.

[0046] More preferably, the antibody fragment according to the present invention is a dsFv fragment of a monoclonal antibody, that is, a disulfide-stabilized Fv fragment in which the heavy chain variable region and the light chain variable region are linked via a disulfide bond. According to one specific embodiment of the present invention, the dsFv fragment is a heterodimer consisting of a heavy chain variable region and a light chain variable region linked via a disulfide bond.

[0047] In yet another aspect, the present disclosure also provides the use of a humanized anti-CD22 antibody in the preparation of a recombinant immunotoxin. For example, to prepare a recombinant immunotoxin, any chain of the humanized anti-CD22 antibody can be linked to the cytotoxin of the present invention.

[0048] The present invention provides a novel humanized anti-CD22 recombinant immunotoxin composed of a humanized anti-CD22 monoclonal antibody fragment fused to a truncated Pseudomonas exotoxin A, specifically a heterodimer composed of a heavy chain variable region and a light chain variable region linked via a disulfide bond, wherein the heavy chain variable region is fused to the truncated Pseudomonas exotoxin A.

[0049] Compared to pre-humanized recombinant immunotoxins, the humanized anti-CD22 recombinant immunotoxin according to the present invention is more similar to that of human counterparts because it is obtained through a specific humanization strategy. Therefore, the immunogenicity of the Fv portion of the immunotoxin in humans is potentially reduced. The reduction in immunogenicity can prevent the generation of unwanted neutralizing anti-drug antibodies (ADA) when used clinically and contribute to a better pharmacokinetic profile.

[0050] Furthermore, the humanized anti-CD22 recombinant immunotoxin according to the present invention maintains the highest affinity for the target CD22 and the corresponding biological activity while having the maximum humanization. Compared to pre-humanized recombinant immunotoxins, the humanized anti-CD22 recombinant immunotoxin of the present invention maintains the binding affinity for the receptor CD22, as well as the cell growth inhibitory effect and apoptosis effect on effector cells. Furthermore, the humanized anti-CD22 recombinant immunotoxin exhibits pharmacokinetics and tumor suppression efficacy comparable to those of pre-engineering ones. [Humanized anti-CD22 recombinant immunotoxin] A humanized anti-CD22 recombinant immunotoxin, which is a polypeptide molecule comprising the following two polypeptide chains: (1) A first polypeptide chain comprising the variable light chain (VL) of an anti-CD22 antibody, wherein the variable light chain (VL) comprises the amino acid sequence shown in SEQ ID NO.16 or SEQ ID NO.18, or an amino acid sequence homolog thereof having at least 75% sequence identity with the amino acid sequence shown in SEQ ID NO.16 or SEQ ID NO.18; (2) A second polypeptide chain comprising the variable heavy chain (V H ) of an anti-CD22 antibody and a cytotoxic agent directly or indirectly linked to the variable heavy chain (V H ), wherein the variable heavy chain (V H ) comprises the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4, or an amino acid sequence homolog thereof having at least 75% identity with the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4. [Humanized anti-CD22 recombinant immunotoxin] The amino acid sequence homolog of the first polypeptide chain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO.16 or SEQ ID NO.18; preferably, the amino acid sequence homolog comprises the amino acid sequences shown in SEQ ID NO.31, SEQ ID NO.32, and SEQ ID NO.33, and has the same amino acid residues as SEQ ID NO.16 or SEQ ID NO.18 at positions 3, 5, 36, 37, 47, 48, 49, 50, 65, 67, 69, 70, and 72 corresponding to the amino acid sequence shown in SEQ ID NO.14, but is not the amino acid sequence shown in SEQ ID NO.14; Preferably, the amino acid sequence homolog of the second polypeptide chain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4; preferably, the amino acid sequence homolog contains the amino acid sequences shown in SEQ ID NO.28, SEQ ID NO.29, and SEQ ID NO.30, and has the same amino acid residues as SEQ ID NO.3 or SEQ ID NO.4 at positions 3, 48, 49, 50, 69, 71, 73, 75, and 80 corresponding to the amino acid sequence shown in SEQ ID NO.2, but is not the amino acid sequence shown in SEQ ID NO.2, the humanized anti-CD22 recombinant immunotoxin of the present invention 1001. [The present invention 1003] The light chain variable region (V L ) of the first polypeptide chain contains the amino acid sequence shown in SEQ ID NO.16 or its amino acid sequence homolog; the heavy chain variable region (VH ) of the second polypeptide chain contains the amino acid sequence shown in SEQ ID NO.3 or its amino acid sequence homolog; or The light chain variable region (V L ) of the first polypeptide chain contains the amino acid sequence shown in SEQ ID NO.18 or its amino acid sequence homolog; the heavy chain variable region (VH) of the second polypeptide chain contains the amino acid sequence shown in SEQ ID NO.3 or its amino acid sequence homolog; or The light chain variable region (V L ) of the first polypeptide chain contains the amino acid sequence shown in SEQ ID NO.16 or its amino acid sequence homolog; the heavy chain variable region (V H ) of the second polypeptide chain contains the amino acid sequence shown in SEQ ID NO.4 or its amino acid sequence homolog; Preferably, in the recombinant immunotoxin, the light chain variable region (V L ) of the first polypeptide chain and the heavy chain variable region (V H ) of the second polypeptide chain are covalently linked, for example, via a disulfide bond; Preferably, the cytotoxin of the second polypeptide chain is Pseudomonas exotoxin A, or a mutant or fragment of Pseudomonas exotoxin A retaining cytotoxicity; preferably, the mutant or fragment of Pseudomonas exotoxin A is PE40, PE38, PE35, PE24, mPE24, T19, T20, or M11, the humanized anti-CD22 recombinant immunotoxin of the present invention 1001 or 1002. [The present invention 1004] In the second polypeptide chain, the cytotoxin is fused to the C-terminus of the variable heavy region (V H ) of the anti-CD22 antibody; Preferably, the N-terminus of the cytotoxin is fused to the C-terminus of the variable heavy region (V H ) directly or via a linker; Preferably, the first polypeptide chain further comprises a κ light chain constant region fused to the C-terminus of the variable light region (V L ), preferably a human κ light chain constant region; or alternatively, the second polypeptide chain further comprises a heavy chain constant region CH1 fused to the C-terminus of the variable heavy region (V H ), preferably a human heavy chain constant region CH1, the humanized anti-CD22 recombinant immunotoxin of any one of the present inventions 1001 to 1003. [The present invention 1005] The first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 16 or an amino acid sequence homolog thereof, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 9 or an amino acid sequence homolog thereof; or The first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 18 or an amino acid sequence homolog thereof, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 9 or an amino acid sequence homolog thereof; or The first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 16 or an amino acid sequence homolog thereof, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 10 or an amino acid sequence homolog thereof, the humanized anti-CD22 recombinant immunotoxin of any one of the present inventions 1001 to 1004. [The present invention 1006] A nucleic acid molecule comprising a nucleotide sequence encoding the first polypeptide chain of the humanized anti-CD22 recombinant immunotoxin of any one of the present inventions 1001 to 1005, and / or a nucleotide sequence encoding the second polypeptide chain of the humanized anti-CD22 recombinant immunotoxin of any one of the present inventions 1001 to 1005. [The present invention 1007] A vector comprising the nucleic acid molecule of the present invention 1006. [The present invention 1008] A host cell transformed or transfected with the nucleic acid molecule of the present invention 1006 or the vector of the present invention 1007. [The present invention 1009] A composition comprising a humanized anti-CD22 recombinant immunotoxin according to any one of the present inventions 1001 to 1005. [The present invention 1010] Use of a humanized anti-CD22 recombinant immunotoxin according to any one of the present inventions 1001 to 1005, the nucleic acid molecule of the present invention 1006, the vector of the present invention 1007, the host cell of the present invention 1008, or the composition of the present invention 1009 in the manufacture of a medicament for the treatment of CD22-related B cell malignancies, Preferably, the CD22-related B cell malignancy is a B cell malignancy characterized by high CD22 expression, such as a lymphoma or leukemia having high CD22 expression; more preferably, the lymphoma is non-Hodgkin lymphoma, small lymphocyte lymphoma, or mantle cell lymphoma; and the leukemia is chronic lymphocytic leukemia, hairy cell leukemia, or acute lymphocytic leukemia. [The present invention 1011] A method for treating CD22-related B cell malignancies, comprising the step of administering to a subject in need thereof a humanized anti-CD22 recombinant immunotoxin according to any one of the present inventions 1001 to 1005, the nucleic acid molecule of the present invention 1006, the vector of the present invention 1007, the host cell of the present invention 1008, or the composition of the present invention 1009, Preferably, the CD22-related B cell malignancy is a B cell malignancy characterized by high CD22 expression, such as a lymphoma or leukemia having high CD22 expression; more preferably, the lymphoma is non-Hodgkin lymphoma, small lymphocyte lymphoma, or mantle cell lymphoma; the leukemia is chronic lymphocytic leukemia, hairy cell leukemia, or acute lymphocytic leukemia; preferably, the subject is a mammal, such as a human or non-human primate, and a breeding animal, livestock, or experimental mammal, such as a dog, cat, cow, pig, sheep, horse, mouse, and rabbit; more preferably, the subject is a human. [The present invention 1012] A humanized anti-CD22 antibody or antibody fragment comprising a heavy chain variable region (V H) and a light chain variable region (V L ), The heavy chain variable region (V H ) comprises the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4, or an amino acid sequence homolog thereof having at least 75% identity with the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4, and the light chain variable region (V L ) comprises the amino acid sequence shown in SEQ ID NO.16 or SEQ ID NO.18, or an amino acid sequence homolog thereof having at least 75% identity with the amino acid sequence shown in SEQ ID NO.16 or SEQ ID NO.18, a humanized anti-CD22 antibody or antibody fragment.

Brief Description of the Drawings

[0051] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0052]

Figure 1

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Figure 3A

Figure 3B

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Figure 5

Modes for Carrying Out the Invention

[0053] Detailed Description of Preferred Embodiments As introduced in the present disclosure, the Fv region of the anti-CD22 portion of HA22 was optimized to be more similar to the Fv region of its human counterpart, and thus a humanization strategy was utilized to potentially reduce the immunogenicity of the Fv portion of the immunotoxin in humans. The reduction in immunogenicity can prevent the generation of unwanted neutralizing anti-drug antibodies (ADA) when used clinically and contribute to a better pharmacokinetic profile.

[0054] Accordingly, the present disclosure provides a humanized anti-CD22 recombinant immunotoxin comprising a humanized anti-CD22 monoclonal antibody fragment fused to a truncated Pseudomonas exotoxin A. Specifically, the humanized anti-CD22 recombinant immunotoxin is a heterodimer composed of a κ light chain V region (first polypeptide chain) linked by one disulfide bond and a heavy chain V region (second polypeptide chain) fused to a truncated Pseudomonas exotoxin A. A schematic diagram of the recombinant immunotoxin is shown in FIG. 1, in which the truncated Pseudomonas exotoxin A is, by way of example, PE38.

[0055] According to the present invention, the humanized anti-CD22 recombinant immunotoxin binds to the CD22 receptor on the surface of malignant B cells and delivers the toxin moiety, e.g., PE38, directly to tumor cells. After internalization, the toxin moiety catalyzes the ADP-ribosylation of the diphthamide residue of elongation factor 2 (EF-2), leading to a rapid decrease in the level of the anti-apoptotic protein, myeloid cell leukemia sequence 1 (Mcl-1), and causing apoptotic cell death.

[0056] The humanized anti-CD22 recombinant immunotoxin according to the present invention can be produced, for example, in an enhanced BL21 derivative (T7 Express) containing an expression plasmid having a nucleotide sequence encoding the first polypeptide chain and an expression plasmid having a nucleotide sequence encoding the second polypeptide chain. The resulting strain is kanamycin resistant, and the production of the humanized anti-CD22 recombinant immunotoxin is induced by isopropyl β-D-1-thiogalactopyranoside (IPTG). The product is expressed as inclusion bodies. The inclusion bodies can be recovered and then refolded in vitro so that heterodimers are formed. The refolded protein can then be purified by hydrophobic interaction chromatography followed by anion exchange chromatography. Finally, the purified protein can be concentrated and diafiltered against a buffer prepared for storage.

[0057] The humanized anti-CD22 recombinant immunotoxin according to the present invention is a humanized version of HA22 and is thus expected to interact with CD22-expressing cells via the same / similar mechanism of action. In vitro and in vivo assays were designed to evaluate the CD22 binding affinity, cytotoxicity in CD22-expressing cells, pharmacokinetics, and tumor inhibitory efficacy of the immunotoxin molecule. HA22 used in the study was manufactured in-house using the same production process as the recombinant immunotoxin.

[0058] In the present disclosure, the interaction between the antibody fragment of the humanized anti-CD22 recombinant immunotoxin of the present invention and the CD22 receptor was measured in an in vitro binding assay; the growth inhibitory effect and apoptotic cell death effect of the humanized anti-CD22 recombinant immunotoxin were investigated in a cell-based assay. Furthermore, the pharmacokinetics and tumor inhibitory efficacy of the humanized anti-CD22 recombinant immunotoxin of the present invention were determined in the in vivo study conducted.

[0059] The sequences used in the present disclosure are as follows.

[0060] SEQ ID NO.1: PE38 polypeptide sequence TIFF0007696648000001.tif42162

[0061] SEQ ID NO.2: HA22, VH polypeptide sequence (HA22 V H ) TIFF0007696648000002.tif11162

[0062] SEQ ID NO.3: Version 1 VH polypeptide sequence (V H Version 1) TIFF0007696648000003.tif11162

[0063] SEQ ID NO.4: Version 2 VH polypeptide sequence (V H Version 2) TIFF0007696648000004.tif11162

[0064] SEQ ID NO.5: Version 3 VH polypeptide sequence (V H Version 3) TIFF0007696648000005.tif12162

[0065] SEQ ID NO.6: Version 4 VH polypeptide sequence (V H Version 4) TIFF0007696648000006.tif12162

[0066] SEQ ID NO.7: Version 5 VH polypeptide sequence (V H Version 5) TIFF0007696648000007.tif12162

[0067] SEQ ID NO.8: HA22 VH-PE38 polypeptide sequence TIFF0007696648000008.tif50162

[0068] SEQ ID NO.9: Version 1 VH-PE38 polypeptide sequence TIFF0007696648000009.tif50162

[0069] SEQ ID NO.10: Version 2 VH-PE38 polypeptide sequence TIFF0007696648000010.tif50162

[0070] SEQ ID NO.11: Version 3 VH-PE38 polypeptide sequence TIFF0007696648000011.tif51162

[0071] SEQ ID NO.12: Version 4 VH-PE38 polypeptide sequence TIFF0007696648000012.tif50162

[0072] SEQ ID NO.13: Version 5 VH-PE38 Polypeptide Sequence TIFF0007696648000013.tif50162

[0073] SEQ ID NO.14: HA22, VL Polypeptide Sequence (HA22 V L ) TIFF0007696648000014.tif12162

[0074] SEQ ID NO.15: Version 1 VL Polypeptide Sequence (V L Version 1) TIFF0007696648000015.tif12162

[0075] SEQ ID NO.16: Version 2 VL Polypeptide Sequence (V L Version 2) TIFF0007696648000016.tif12162

[0076] SEQ ID NO.17: Version 3 VL Polypeptide Sequence (V L Version 3) TIFF0007696648000017.tif12162

[0077] SEQ ID NO.18: Version 4 VL Polypeptide Sequence (V L Version 4) TIFF0007696648000018.tif11162

[0078] SEQ ID NO.19: Version 1 VH-PE38 DNA Sequence TIFF0007696648000019.tif159151

[0079] SEQ ID NO.20: Version 2 VH-PE38 DNA Sequence TIFF0007696648000020.tif159151

[0080] SEQ ID NO.21: Version 3 VH-PE38 DNA sequence TIFF0007696648000021.tif158151

[0081] SEQ ID NO.22: Version 4 VH-PE38 DNA sequence TIFF0007696648000022.tif159151

[0082] SEQ ID NO.23: Version 5 VH-PE38 DNA sequence TIFF0007696648000023.tif159151

[0083] SEQ ID NO.24: Version 1 VL DNA sequence TIFF0007696648000024.tif34151

[0084] SEQ ID NO.25: Version 2 VL DNA sequence TIFF0007696648000025.tif34151

[0085] SEQ ID NO.26: Version 3 VL DNA sequence TIFF0007696648000026.tif35151

[0086] SEQ ID NO.27: Version 3 VL DNA sequence TIFF0007696648000027.tif35151

[0087] SEQ ID NO.28: HA22, H-CDR1 GFAFSIYD

[0088] SEQ ID NO.29: HA22, H-CDR2 ISSGGGTTY

[0089] SEQ ID NO.30: HA22, H-CDR3 CARHSGYGTHWGVLFAY

[0090] SEQ ID NO.31: HA22, L-CDR1 QDISNY

[0091] SEQ ID NO.32: HA22, L-CDR2 YTSILHSG

[0092] SEQ ID NO.33: HA22, L-CDR3 QQGNTLP

Example

[0093] The present invention will be described below with reference to specific examples. It will be understood by those skilled in the art that these examples are merely illustrative of the present invention and in no way limit the scope of the present invention.

[0094] Unless otherwise specified, all experimental procedures in the following examples are conventional. Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available products.

[0095] Example 1: Humanization Design Heavy chain variable region (V H , SEQ ID NO.2) and light chain variable region (V L, the amino acids in the framework regions of SEQ ID NO. 14 were modified from those of the original mouse to those of human using a framework shuffling strategy. The CDRs determined using both the Kabat and Chothia numbering systems as well as the online tool (http: / / www.bioinf.org.uk / abs / ) were all left unchanged. Each of the framework regions and J regions in both VH and VL of HA22 was aligned with the human antibody germline sequences of the international ImMunoGeneTics information system (registered trademark) (IMGT: http: / / www.imgt.org) for the closest human antibody match. Mouse residues considered important for Vernier residues or VH / VL interactions were not changed in order to preserve the specificity and affinity for CD22.

[26] .

[0096] To select the final candidates with maximum humanization while maintaining optimal affinity and potency profiles, a panel of humanized designs was prepared and evaluated. The improved VH and VL formed a disulfide-linked Fv portion of an anti-CD22 antibody fused with Pseudomonas exotoxin A (PE38, SEQ ID NO. 1) (Figure 1).

[0097] Alignment revealed that the combination of human germline sequences, framework 1 of IGHV3-15*01, framework 2 of IGHV3-11*04, framework 3 of IGHV3-13*01, and framework 4 of IGHJ3*02 provided the most similar humanized framework to the VH framework of HA22; similarly, the combination of framework 1 of IGKV1-5*01, framework 2 of IGKV2D-30*01, framework 3 of IGKV1D-43*01, and framework 4 of IGKJ1*01 provided the most similar humanized framework to the VL framework of HA22. The Vernier residues were reverted to the original mouse residues after several iterations to show that they were important for antigen binding.

[0098] The sequence alignment provided in FIG. 2 shows an example of the humanization process. In the alignment, HA22VH and HA22VL are the Fv fragments used in HA22. Version 3 (V3) is the fully humanized version of VH and VL. Versions 1, 2, 4, and 5 (V1, V2, V4, and V5) are different versions of the design in which important residues were reverted to mouse residues to restore the lost activity. The shaded residues are the Burnier residues. The underlined residues are within the CDRs based on the IMGT criteria.

[0099] Example 2: Preparation of Humanized Anti-CD22 Recombinant Immunotoxin Expression plasmids capable of expressing different VL humanized versions (first polypeptide chain) were constructed. Further, the 5' end of the nucleotide sequence encoding PE38 was ligated to the 3' end of each of the nucleotide sequences encoding different VH humanized versions to form a single nucleotide chain, thereby constructing an expression plasmid capable of expressing the second polypeptide chain. A schematic diagram is shown in FIG. 3 (synthesized and constructed by Genscript according to the design).

[0100] The humanized anti-CD22 recombinant immunotoxin provided in the present disclosure was produced in an enhanced BL21 derivative strain (T7 Express) using the expression plasmids of the first and second polypeptide chains.

[0101] 1. Fermentation Seed cultures of the first polypeptide chain and the second polypeptide chain were each prepared in a fermentation medium containing 30 g of soytone, 30 g of yeast extract, 40 g of glycerol, 2 g of NH4Cl, 2 g of (NH4)2SO4, 0.973 g of MgSO4, 3 g of glucose, and 1 g of NaCl per liter. The seed cultures were incubated at 37 °C and 250 RPM for 12 - 16 hours. Fermentation runs for the first polypeptide chain and the second polypeptide chain were carried out using a New Brunswick BioFlo 3000 fermenter. The fermenter was seeded with 2% of each of the seeds of the first polypeptide chain and the second polypeptide chain. The pH was set at 6.9 and controlled during the run by the addition of ammonia (NH3·H2O). Dissolved oxygen (DO) was set at 30% and controlled by stirring, aeration, and oxygen supplementation. The vessel pressure was set at 2 psi. Cell density (OD 600 ) and glucose concentration were monitored every hour during the run, along with pH, DO, and temperature. Four hours after inoculation, when OD 600 was 8 - 9, the glucose in the medium was depleted and additional glucose was fed to the fermenter. The glucose feed was continued for 1 hour and the total volume was 1.7% of the initial medium volume. The additional glucose was consumed approximately 5.5 hours after inoculation. Then, 0.1% volume of 1 M IPTG was added to initiate the induction / expression phase. During the induction period, the pH was gradually raised to 7.0. Three hours after induction, the cells were harvested by centrifugation at 8,000×g and 4 °C for 20 minutes to remove the medium. The cell paste was stored at -80 °C before processing.

[0102] 2. Recovery and refolding Inclusion bodies of the first polypeptide chain and the second polypeptide chain were isolated by disrupting the cells using a high-pressure homogenizer. The lysate pellet containing the inclusion bodies was then purified through several washing steps with centrifugation and surfactant to remove cell debris and soluble impurities.

[0103] To maintain all cysteines in a reduced state and cleave the disulfide bonds formed during preparation, the washed inclusion bodies were resuspended in a solubilization buffer (50 mM ethanolamine, 8 M urea, 0.5 M arginine, 2 mM EDTA, 10 mM DTT, pH 9.3) containing a strong denaturing agent (8 M urea) and a reducing agent (10 mM DTT). Solubilization was carried out at 2 - 8 °C with stirring to facilitate a solubilization process exceeding 2 hours. After solubilization, to remove the remaining aggregates and insoluble impurities, the inclusion body solution was centrifuged at 13,000×g for 30 minutes, and the supernatant containing the solubilized inclusion bodies was filtered.

[0104] The total protein concentration was determined based on the reading of A280, and the percentages of the first polypeptide chain and the second polypeptide chain were estimated by densitometric analysis of the SDS-PAGE gel. Then, the first polypeptide chain and the second polypeptide chain were mixed at a molar ratio of 1:1, diluted to 10 mg / mL with the solubilization buffer, and continuously fed at a ratio of 1:10 into a refolding buffer (50 mM ethanolamine, 0.5 M arginine, 2 mM EDTA, 0.9 mM oxidized glutathione, pH 9.4) for 24 hours, and further incubated at 2 - 8 °C for 48 - 72 hours. The refolding reaction was stopped by adjusting the pH to approximately 7.40. Then, the refolded protein was concentrated and prepared for column purification by tangential flow filtration.

[0105] 3. Phenyl HP Hydrophobic Interaction Chromatography Phenyl HP hydrophobic interaction chromatography was performed on a chromatography column (GE Healthcare, Marlborough, MA USA) with a bench height of 17 cm. All runs were performed using an AKTA avant liquid chromatography system from GE Healthcare (Marlborough, MA USA), and the column was operated at 95 cm / hr. The column was equilibrated with 20 mM Tris-HCl, 0.6 M Na2SO4, pH 7.4. A loading solution was prepared by diluting 1 part of the protein solution with 1 part of 20 mM Tris-HCl, 1.2 M Na2SO4, pH 7.4. After loading, the column was washed with the equilibration buffer and then eluted over 16 column volumes with a linear gradient of Na2SO4 from 0.6 M to 0 M. The product peak was collected in fractions.

[0106] 4. POROS HQ Anion Exchange Chromatography POROS HQ anion exchange chromatography was performed on a chromatography column (Thermo Scientific, Waltham, MA USA) packed to a bed height of 30 cm. All runs were performed using an AKTA avant liquid chromatography system from GE Healthcare, and the column was operated at 275 cm / hr. The column was equilibrated with 20 mM Tris-HCl, pH 7.4, the protein was loaded, and then the column was washed with the equilibration buffer. The column was eluted over 25 column volumes with a linear gradient of NaCl from 0 M to 0.5 M. The product peak was collected based on an absorbance criterion of 5 mAU at the leading and trailing sides of the product peak.

[0107] 5. Capto Q Anion Exchange Chromatography In a chromatography column (GE Healthcare, Marlborough, MA USA) filled with a bed height of 10 cm, Capto Q anion exchange chromatography was performed in flow-through mode. All runs were performed using a GE Healthcare AKTA avant liquid chromatography system, and the column was operated at 250 - 350 cm / hr. The column was equilibrated with 20 mM Tris-HCl, 250 mM NaCl, pH 7.4, the protein was loaded, and then washed with the equilibration buffer. Product peaks were collected based on an absorbance criterion of 5 mAU on the leading and trailing sides of the product flow-through peak.

[0108] Example 3: Non-clinical Pharmacology of Humanized Anti-CD22 Recombinant Immunotoxin As described in Example 2, various recombinant immunotoxins were prepared using various humanized versions of VH and VL provided in the present disclosure, as well as VH and VL of HA22, and named according to the format "HmLn" (m and n are the numbers of the humanized versions of VH and VL).

[0109] 3.1 Target Interaction ELISA was performed using recombinant human Siglec-2 / CD22 Fc chimera protein (R&D systems, catalog number 1968-SL) as an immobilized antigen to capture the humanized anti-CD22 recombinant immunotoxin or HA22 molecule provided in the present disclosure, and anti-Pseudomonas exotoxin A antibody (Sigma-Aldrich, catalog number P2318) as a secondary antibody containing an enzyme conjugate for detection.

[0110] The K of the recombinant immunotoxin or HA22 molecule provided in the present disclosure D For measurement, an Octet assay was performed using a streptavidin (SA) biosensor (ForteBio, catalog number 18-5019) conjugated with biotinylated CD22.

[0111] As shown in Table 1, (V H version 1 + V L version 2-containing) recombinant immunotoxin H1L2 and (V H version 2 + V L version 2-containing) H2L2 showed EC 50 and K D comparable to HA22.

[0112] (Table 1) CD22 binding assay results TIFF0007696648000028.tif32141

[0113] Different versions of the humanized recombinant immunotoxin were analyzed for CD22 binding affinity as shown in Tables 2 and 3. In Table 2, the range of EC 50 is given or presented as the mean ± SD when two or more batches of the product were assayed separately in different analyses. The results of EC 50 of the humanized recombinant immunotoxin compared to HA22 are shown in Table 3.

[0114] (Table 2) CD22 binding affinity (nM) TIFF0007696648000029.tif56170

[0115] (Table 3) CD22 binding affinity (EC 50 normalized to HA22) TIFF0007696648000030.tif59170

[0116] 3.2 Cell-based titer assay To evaluate the growth inhibitory effect and apoptotic cell death effect of the humanized anti-CD22 recombinant immunotoxin in the present disclosure, a CD22-expressing cell model was utilized in a cell-based assay. This is an endpoint assay that uses cell growth / inhibition as an indicator of titer. The cell model better mimics in vivo conditions in which CD22 binding, internalization, and ADP ribosylation of EF-2 catalyzed by PE38 are all involved in exerting the observed inhibitory effect, as compared to the binding assay.

[0117] In this assay, the Daudi cell line (ATCC) that expresses high levels of CD22 was co-cultured with the recombinant immunotoxin or HA22 molecule provided in the present disclosure for a certain period of time, and at the end of the assay, the viable cells in the culture were quantified by a colorimetric assay. The procedure is as follows.

[0118] The above-mentioned Daudi cells were cultured in RPMI-1640 complete medium, collected, and washed once with HBSS. The cells were resuspended in RPMI-1640 medium at a density of 2.5×10 5 / mL and transferred to a 96-well plate at 100 μL / well (equivalent to 2.5×10 4 viable cells per well). The recombinant immunotoxin or HA22 provided in the present disclosure to be detected was diluted to a predetermined concentration, and serial dilutions were prepared.

[0119] 100 μL of each dilution was added to the cells in the well, and the cells were incubated at 37°C and 5% CO2 for 48 to 72 hours. Then, 10 μL of the CCK-8 reagent was added to each well of the plate, and then it was incubated at 37°C for 4 to 8 hours. To correct for background activity, the cells were cultured in the presence of 10 μg / mL cycloheximide until 100% of the cells were dead.

[0120] The production of formazan was measured by detecting the absorbance at 450 nm. The values were normalized against cycloheximide and media controls, and the concentration of each of the humanized recombinant immunotoxins and HA22 provided in the present disclosure at which 50% of the cells were killed was measured as the EC 50 .

[0121] Various versions of the humanized recombinant immunotoxins were analyzed for CD22 binding affinity as shown in Tables 4 and 5. In Table 4, the range of EC 50 is given or presented as the mean ± SD when two or more batches of the product were assayed separately in separate analyses. The results of the EC 50 of the humanized recombinant immunotoxins compared to HA22 are shown in Table 5. As shown, the humanized recombinant immunotoxins, particularly H1L2, showed EC 50 comparable to HA22 in in vitro cell-based potency assays.

[0122] (Table 4) In vitro potency assay (pM) TIFF0007696648000031.tif55170

[0123] (Table 5) In vitro potency assay (EC 50 normalized against HA22) TIFF0007696648000032.tif60170

[0124] Example 4: Non-clinical Pharmacokinetics and Metabolism of Humanized Anti-CD22 Recombinant Immunotoxins 4.1 In Vivo PK Study In this study, C57BL / 6 mouse models (4 - 6 weeks old, female) were used. Forty-five mice were randomly divided into three groups: (1) control (formulation buffer), (2) 500 μg / kg HA22; and (3) 500 μg / kg H1L2. The control or treatment groups were given a tail vein injection, and at the designated time points after injection (5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours, 96 hours), 100 μL of whole blood was collected via retro-orbital sampling. Three replicate animals were assigned to each time point. For analysis, 50 μL of plasma was recovered from the blood samples.

[0125] As shown in Tables 6 and 7, the results showed that the mouse model H1L2 exhibited pharmacokinetic properties comparable to HA22: both molecules had similar half-lives (T 1 / 2 ), time to reach maximum concentration (T max ), maximum concentration (C max ), and area under the curve (AUC 0-t ). The PK profiles are shown in Figure 4.

[0126] (Table 6) HA22 PK Results TIFF0007696648000033.tif44166

[0127] (Table 7) H1L2 PK Results TIFF0007696648000034.tif44166

[0128] 4.2 In Vivo Efficacy Xenograft animal models and CD22-positive Raji B lymphocytes were utilized for tumor establishment. After administration, the recombinant immunotoxin molecules entered the circulatory system, bound to CD22 on the surface of malignant B cells, and suppressed cell proliferation via the apoptotic pathway. Therefore, inhibition of tumor growth was designated as the endpoint of the efficacy study in this example.

[0129] The thymus-deficient NCr nude mouse model (4 - 6 weeks old, female) was used. Twenty-five mice were randomly divided into five groups as shown in Table 8. The CD-22 positive Homo sapiens lymphoblast Raji cell line (ATCC CCL-86) was used to establish xenografts. Raji cells were subcutaneously injected at a final concentration of 5×10 7 cells / mL and allowed to grow to 100 - 200 mm 3 before treatment. After the xenograft tumors were established, treatment at the indicated dose levels was administered as a single injection via the tail vein. The xenograft mice were observed for 24 days after treatment, and the tumor size was measured.

[0130] As shown in Figure 5, compared with the control group (Group 1), the high-dose HA22 group showed an 84.9% inhibition of tumor growth, and the low-dose group showed a 95.8% inhibition of tumor growth; the high-dose H1L2 group showed an 87.7% inhibition of tumor growth, and the low-dose group showed a 34.2% inhibition of tumor growth. When a t-test was performed, all treatments showed significant tumor suppression efficacy except for the low-dose humanized recombinant immunotoxin H1L2 (in Figure 5, * P < 0.05, ** P < 0.01, *** P < 0.005). There was no statistically significant difference between the high-dose HA22 group and the high-dose H1L2 group.

[0131] (Table 8) Groups and dosing information for the efficacy study TIFF0007696648000035.tif48166

[0132] The foregoing description of the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art can make various changes and modifications to the present invention without departing from the spirit of the present invention, and these shall be included in the scope of the appended patent claims.

[0133] References: TIFF0007696648000036.tif14163TIFF0007696648000037.tif241168TIFF0007696648000038.tif242163TIFF0007696648000039.tif64163

Claims

1. A humanized anti-CD22 recombinant immunotoxin, which is a polypeptide molecule comprising the following two polypeptide chains: (1) The light chain variable region (V L ) of an anti-CD22 antibody comprising the amino acid sequence shown in SEQ ID NO. 16, a first polypeptide chain; (2) The heavy chain variable region (V H ) of an anti-CD22 antibody comprising the amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4, and a cytotoxic agent which is a mutant or fragment of Pseudomonas exotoxin A or Pseudomonas exotoxin A retaining cytotoxicity, wherein the N-terminus of the cytotoxic agent is directly or via a linker fused to the C-terminus of the heavy chain variable region (VH), a second polypeptide chain.

2. In the recombinant immunotoxin, the light chain variable region (V L ) of the first polypeptide chain and the heavy chain variable region (V H ) of the second polypeptide chain are covalently linked, the humanized anti-CD22 recombinant immunotoxin according to Claim 1.

3. The light chain variable region (VL) of the first polypeptide chain and the heavy chain variable region (VH) of the second polypeptide chain are covalently linked via a disulfide bond, the humanized anti-CD22 recombinant immunotoxin according to Claim 1.

4. The mutant or fragment of Pseudomonas exotoxin A is PE40, PE38, PE35, PE24, mPE24, T19, T20, or M11, the humanized anti-CD22 recombinant immunotoxin according to any one of Claims 1 to 3.

5. The first polypeptide chain further comprises a κ light chain constant region fused to the C-terminus of the light chain variable region (V L ), the humanized anti-CD22 recombinant immunotoxin according to any one of Claims 1 to 4.

6. The κ light chain constant region is a human κ light chain constant region, the humanized anti-CD22 recombinant immunotoxin according to Claim 5. **Claim 7**: The humanized anti-CD22 recombinant immunotoxin according to any one of claims 1 to 6, further comprising a heavy chain constant region CH1 to which a second polypeptide chain is fused at the C-terminus of the heavy chain variable region (VH). **Claim 8**: The humanized anti-CD22 recombinant immunotoxin according to claim 7, wherein the heavy chain constant region CH1 is a human heavy chain constant region CH1. **Claim 9** The first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 16, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 9; or The first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 16, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 10, the humanized anti-CD22 recombinant immunotoxin according to any one of claims 1 to 4. **Claim 10** A nucleic acid molecule comprising a nucleotide sequence encoding the first polypeptide chain of the humanized anti-CD22 recombinant immunotoxin according to any one of claims 1 to 9, and a nucleotide sequence encoding the second polypeptide chain of the humanized anti-CD22 recombinant immunotoxin according to any one of claims 1 to 9. **Claim 11** A vector comprising the nucleic acid molecule according to claim 10. **Claim 12** A host cell transformed or transfected with the nucleic acid molecule according to claim 10 or the vector according to claim 11. **Claim 13** A composition comprising the humanized anti-CD22 recombinant immunotoxin according to any one of claims 1 to 9. **Claim 14** Use of the humanized anti-CD22 recombinant immunotoxin according to any one of claims 1 to 9, the nucleic acid molecule according to claim 10, the vector according to claim 11, the host cell according to claim 12, or the composition according to claim 13 in the manufacture of a medicament for the treatment of CD22-related B cell malignancies.

15. The use according to claim 14, wherein the CD22-related B cell malignancy is a B cell malignancy characterized by high CD22 expression.

16. The use according to claim 14 or 15, wherein the CD22-related B cell malignancy is a lymphoma or leukemia having high CD22 expression.

17. The use according to claim 16, wherein the lymphoma is non-Hodgkin lymphoma, small lymphocyte lymphoma, or mantle cell lymphoma; and the leukemia is chronic lymphocytic leukemia, hairy cell leukemia, or acute lymphocytic leukemia.

18. A humanized anti-CD22 antibody or antibody fragment comprising a heavy chain variable region (V H ), and a light chain variable region (V L ), wherein the heavy chain variable region (V H ) comprises the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4, and the light chain variable region (V L ) comprises the amino acid sequence shown in SEQ ID NO.

16.

19. The humanized anti-CD22 antibody or antibody fragment according to claim 18, wherein the humanized anti-CD22 antibody is a monoclonal antibody.

20. The humanized anti-CD22 antibody or antibody fragment according to claim 18 or 19, wherein the antibody fragment is Fab, Fab', F(ab')2, scFv, or dsFv.

21. The humanized anti-CD22 antibody or antibody fragment according to any one of claims 18 to 20, wherein the antibody fragment is a dsFv fragment.

22. The use of the humanized anti-CD22 antibody or antibody fragment according to any one of claims 18 to 21 in the preparation of the humanized anti-CD22 recombinant immunotoxin according to any one of claims 1 to 9.

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