Humanized antibody against CD79b

Abstract

The present invention relates to humanized antibodies or target-binding fragments or derivatives thereof that retain target-binding ability, which bind to human CD79b (Figure 1).

Description

[Technical Field]

[0001] The present application relates to a humanized antibody against CD79b. [Background technology]

[0002] Despite recent advances in basic treatment, many types of cancer remain difficult or impossible to treat successfully. This is particularly true for B-cell-related malignancies. Targeted therapies exist, particularly against CD20, CD19, and CD22.

[0003] CD79b (immunoglobulin-related beta), another antigen on B cells, has been shown to have advantages for use as a target, particularly over CD20.

[0004] CD79b, together with CD79a and surface immunoglobulins, constitutes the BCR cell receptor, which is expressed in over 90% of B-cell non-Hodgkin's lymphomas. CD79b is internalized upon antibody binding. This functionality may allow CD79b to selectively deliver molecules of interest to B cells found in non-Hodgkin's lymphoma (NHL).

[0005] Antibodies against CD79b have been shown to inhibit B cell proliferation induced through the B cell receptor, CD40, CD180, and chondroitin sulfate, but not through TLR4 or TLR9 (Bruhl et al. 2015).

[0006] Anti-CD20 antibody therapy remains the standard of care for many patients with B-cell malignancies: approximately two-thirds of patients benefit from this approach, while one-third do not.

[0007] Given this background, CD79b may offer a new therapeutic option. In a phase III clinical trial reported by Tilly et al. (2021), 879 patients aged 18 to 80 years with intermediate- to high-risk, poor-prognosis diffuse large B-cell lymphoma received standard treatment with an anti-CD20 antibody plus chemotherapy (R-CHOP) or an antibody-drug conjugate against CD79b plus chemotherapy (pola-R-CHP). Significantly more patients in the pola-R-CHP group survived without further disease progression. Patients who received the antibody-drug conjugate plus chemotherapy had a lower risk of lymphoma progression, recurrence, and death.

[0008] The anti-antibody drug conjugate used in this study is polatuzumab vedotin (Polivy®), also known as DCDS4501A or RG7596.

[0009] Polivy is an antibody-drug conjugate comprising an IgG anti-CD79b antibody and the toxin MMAE, which is composed of para-aminobenzyl carbamate conjugated to the free thiol group of a cysteine ​​residue in a cysteine-engineered antibody ("Thiomab"), a cathepsin-cleavable linker containing citrulline and valine, and a linking group consisting of caproic acid and maleimide, via a linker / spacer. Polivy has an average of 3.5 molecules of MMAE conjugated per antibody, but the drug-to-antibody ratio (DAR) varies.

[0010] The antibody polatuzumab is a humanized variant of the murine anti-CD79b antibody SN8, first described by Okazaki et al. in 1993. Polatuzumab and polatuzumab vedotin are disclosed, inter alia, in EP 2176296 and US 8545850.

[0011] One object of the present invention is to provide new and improved options for the treatment of cancer.

[0012] These and other objects are solved by the features of the independent claims. The dependent claims disclose embodiments of the invention that may be preferred in particular circumstances. Similarly, the specification discloses further embodiments of the invention that may be preferred in particular circumstances. [Brief explanation of the drawings]

[0013] [Figure 1] 1, 2, and 3 show the results of in vitro cell viability assays of four recombinant immunotoxins according to the present invention (IgG antibodies fused to the protein toxin anisopurine via a G4S linker) and a benchmark antibody-drug conjugate (polatuzumab vedotin). It can be seen that the four immunotoxins according to the present invention have significantly better IC50s (in vitro potency) than the benchmark antibody-drug conjugate. [Figure 2] As mentioned above. [Figure 3] As mentioned above. [Figure 4] Figures 4 and 5 show the results of size-exclusion chromatography experiments of a recombinant immunotoxin according to the present invention (an IgG antibody fused via a G4S linker to the protein toxin anisoprine) and a benchmark recombinant immunotoxin (polatuzumab fused via a G4S linker to the protein toxin anisoprine). Figure 4A: ATB 704, Figure 4B: ATB-580. [Figure 5] Figure 5A, column calibration; Figure 5B: benchmark (polatuzumab). The recombinant immunotoxins according to the invention are highly stable, whereas the polatuzumab-based benchmark (ATB-452) shows a higher tendency to aggregate. [Figure 6]Figures 6 and 7 show IHC staining of frozen human tissues from three donors under conditions favoring high-affinity interaction with the cognate receptor (on-target binding). Specific staining with the recombinant immunotoxins ATB-580, ATB-693, ATB-697, and ATB-704 (IgG antibodies fused to the protein toxin anisopurine via a G4S linker) was observed only in CD79b-positive human tissues, such as lymph nodes, spleen, and thymus. Off-target binding to CD79b-negative tissues was not observed for any of the tested compounds. [Figure 7] As mentioned above. [Figure 8] Figure 8 shows a comparison of the in vivo antitumor activity of four recombinant immunotoxins according to the present invention (IgG antibodies fused to the protein toxin anisopurine via a G4S linker) with a benchmark recombinant immunotoxin (polatuzumab fused to the protein toxin anisopurine via a G4S linker, herein referred to as "ATB-747"). For this purpose, a CDX model (transplanted with a representative B-NHL cell line) in scSCID mice was used. [Figure 9] FIG. 9 shows an alignment of the different isoforms of CD79B, with the epitopes discussed herein in bold. DETAILED DESCRIPTION OF THE INVENTION

[0014] (Detailed explanation) According to one aspect of the present invention, there is provided an antibody or a target-binding fragment or derivative thereof that retains target binding ability to human CD79b, comprising: a) SEQ ID NOs: 3 and 4; SEQ ID NOs: 13 and 14, SEQ ID NOs: 23 and 24, or SEQ ID NOs: 33 and 34 or a set of six heavy / light chain complementarity determining regions (CDRs) contained in a heavy chain / light variable domain sequence pair selected from one of the pairs b) In the order of HCDR1;HCDR2;HCDR3;LCDR1;LCDR2 and LCDR3, i) SEQ ID NOs: 5, 6, 7, 8, 9, and 10; ii) SEQ ID NOs: 15, 16, 17, 18, 19, and 20; iii) SEQ ID NOs: 25, 26, 27, 28, 29, and 30, or iv) SEQ ID NOs: 35, 36, 37, 38, 39, and 40 or a set of six heavy / light chain complementarity determining regions (CDRs) selected from c) a set of heavy / light chain complementarity determining regions (CDRs) according to option b), with the proviso that at least one of the CDRs has up to three amino acid substitutions relative to the CDR contained in the respective SEQ ID NO:; and / or d) a set of heavy / light chain complementarity determining regions (CDRs) from option b) or c), with the proviso that at least one of the CDRs has 66% or greater sequence identity to the respective SEQ ID NOs; Herein, there is provided an antibody or a target-binding fragment or derivative thereof that retains target binding ability to human CD79b, wherein the CDRs are embedded in a suitable protein framework so as to be able to bind to human CD79b.

[0015] The following table provides a brief list of antibodies according to the present invention. Antibody names and sequences used herein [Table 1]

[0016] In one embodiment, the antibody is a humanized antibody or fragment.

[0017] Methods for producing and / or selecting humanized mAbs are known in the art, for example, US6331415 to Genentech describes the production of chimeric antibodies, while US6548640 to the Medical Research Council describes CDR grafting technology, and US5859205 to Celltech describes the production of humanized antibodies.

[0018] A humanized antibody is an antibody in which the complementarity-determining regions (CDRs) of a parent antibody from a non-human species are grafted onto the framework (at least the variable domains) of a human antibody, such as IgG1, IgG2, or IgG4. Humanized antibodies bind to the same targets as the parent antibody, but because they are grafted onto a human framework, they are less immunogenic (e.g., HAMA reaction). Therefore, humanized antibodies are structurally distinct from the parent (e.g., murine) antibody.

[0019] In humanization, the step of grafting CDRs onto a human framework is often followed by a step of affinity maturation to regain affinity lost in the grafting process, which further modifies the sequence of the human antibody, including the CDRs.

[0020] As used herein, the term "CDR" or "complementarity determining region" is intended to mean the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These particular regions are described by Kabat et al. (1977), Kabat et al. (1991), Chothia et al. (1987), where the definitions include overlapping or subsets of amino acid residues when compared with each other.

[0021] Nevertheless, application of either definition to refer to an antibody or grafted antibody CDR or variant thereof is intended to be within the scope of the term as defined and used herein.

[0022] Preferably, the CDRs described herein are determined according to the Kabat or Chothia numbering set forth in Table 2.

[0023] CDR definition [Table 2]

[0024] As used herein, the term "framework" when used in reference to an antibody variable region is intended to mean all amino acid residues outside the CDR regions within the variable region of an antibody. Thus, although the framework of a variable region is approximately 100-120 amino acids in length, it is intended to refer only to amino acids outside the CDRs.

[0025] In one embodiment, the term "capable of binding to target X" refers to a binding domain that is 10 -4 The following K D It should be understood to mean that the target binds at K D is the equilibrium dissociation constant, k between an antibody or fragment and its antigen off / k on is the ratio of K D and affinity are inversely related. D The K value is related to the antibody or fragment concentration (the amount of antibody or fragment needed for a particular experiment) and therefore D Lower values ​​(lower concentrations) indicate higher affinity of the binding domain. The following table shows typical K values ​​for monoclonal antibodies: D The range is shown.

[0026] K D and molar value [Table 3]

[0027] Preferably, the antibody or fragment has up to two amino acid substitutions, more preferably up to one amino acid substitution. Preferably, at least one of the CDRs of the antibody or fragment has sequence identity to a respective SEQ ID NO: 67% or more; 68% or more; 69% or more; 70% or more; 71% or more; 72% or more; 73% or more; 74% or more; 75% or more; 76% or more; 77% or more; 78% or more; 79% or more; 80% or more; 81% or more; 82% or more; 83% or more; 84% or more; 85% or more; 86% or more; 87% or more; 88% or more; 89% or more; 90% or more; 91% or more; 92% or more; 93% or more; 94% or more; 95% or more; 96% or more; 97% or more; 98% or more; 99% or more, and most preferably 100%.

[0028] As used herein, "percentage of sequence identity" is determined by comparing two optimally aligned biological sequences (amino acid sequences or polynucleotide sequences) over a comparison window, where the corresponding portions of the sequences in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence that does not contain additions or deletions due to optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0029] The term "identical" or percent "identity," with respect to two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same sequence. Two sequences are "substantially identical" if they contain a specified percentage of the same amino acid residues or nucleotides (i.e., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity over a specified region, or, if not specified, the entire reference sequence) when compared and aligned for maximum correspondence over a comparison window or specified region, measured using one of the sequence comparison algorithms described below, or by manual alignment and visual inspection. The present disclosure provides polypeptides that are substantially identical to the polypeptides exemplified herein. With respect to amino acid sequences, identity or substantial identity can exist over a region at least 5, 10, 15, or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75, or 100 amino acids in length, optionally at least about 150, 200, or 250 amino acids in length, or over the entire length of the reference sequence. For shorter amino acid sequences, for example, sequences of 20 or fewer amino acids, substantial identity exists if one or two amino acid residues are conservatively substituted in accordance with conservative substitutions as defined herein.

[0030] Preferably, at least one of the CDRs comprises: affinity maturation Reduced immunogenicity The CDR sequences were subjected to modifications including:

[0031] Affinity maturation is the process of increasing the affinity of a given antibody in vitro. Similar to its natural counterpart, in vitro affinity maturation is based on the principle of mutation and selection. It has been successfully used to optimize antibodies, antibody fragments, or other peptide molecules such as antibody mimetics. Random mutations within CDRs are introduced using radiation, chemical mutagens, or error-prone PCR. Furthermore, chain shuffling can increase genetic diversity. Two or three rounds of mutation and selection using display methods such as phage display typically result in antibody fragments with affinities in the low nanomolar range. For a discussion of the principles, see Eylenstein et al. (2016) or US20050169925A1, the contents of which are incorporated herein by reference for purposes of enablement.

[0032] The antibody produced comprises the CDR region derived from mouse sequence, together with the necessary framework back mutations in the V region derived from sequence.Therefore, when humanized antibody is administered to a patient, CDR itself can cause immunogenic reaction.Methods for reducing the immunogenicity caused by CDR are disclosed in Harding et al. (2010) or US2014227251A1, the contents of which are incorporated herein by reference for the purpose of implementation.

[0033] According to one embodiment of the invention, the antibody or fragment comprises: a) SEQ ID NOs: 3 and 4; SEQ ID NOs: 13 and 14, SEQ ID NOs: 23 and 24, or A pair of heavy chain / light chain variable domains (HCVD / LCVD) as set forth in the pair of SEQ ID NOs: 33 and 34; b) a pair of heavy chain / light chain variable domains (HCVD / LCVD) of a) with the proviso that: the HCVD has 80% or greater sequence identity to the HCVD contained in the respective SEQ ID NO: and / or LCVDs are pairs that have 80% or more sequence identity to the LCVDs contained in each SEQ ID NO: c) A heavy chain / light chain variable domain (HCVD / LCVD) pair according to option a) or b), wherein at least one of the HCVDs or LCVDs has up to 10 amino acid substitutions relative to the HCVD or LCVD contained in the respective SEQ ID NO:

[0034] It is provided that the antibody or fragment is capable of binding to human CD79b.

[0035] A "variable domain" as used in reference to an antibody or its heavy or light chain is intended to refer to a portion of an antibody that confers antigen binding on the molecule and is not the constant region. The term is intended to include functional fragments thereof that maintain some of the binding function of the entire variable region. Variable region-binding fragments include, for example, functional fragments of Fab, F(ab)2, Fv, single-chain Fv (scfv), etc. Such functional fragments are well known to those skilled in the art. Thus, the use of these terms in describing functional fragments of heteromeric variable regions is intended to correspond to definitions well known to those skilled in the art. Such terms are described, for example, in Huston et al. (1993) or Plückthun and Skerra (1990).

[0036] Preferably, the HCVD and / or LCVD have 81% or more; 82% or more; 83% or more; 84% or more; 85% or more; 86% or more; 87%; 88% or more; 89% or more; 90% or more; 91% or more; 92% or more; 93%; 94% or more; 95% or more; 96% or more; 97% or more; 98% or more; 99% or more; or most preferably 100% sequence identity to their respective SEQ ID NOs.

[0037] According to one embodiment of the present invention, at least one amino acid substitution is a conservative amino acid substitution.

[0038] As used herein, "conservative amino acid substitutions" have a smaller effect on antibody function than non-conservative substitutions. Although there are various ways to classify amino acids, they are often divided into six major groups based on their structure and the general chemical properties of the R groups.

[0039] In some embodiments, a "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. For example, families of amino acid residues having similar side chains have been defined in the art. These families include: Basic side chains (lysine, arginine, histidine, etc.), acidic side chains (aspartic acid, glutamic acid, etc.), Uncharged polar side chains (glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, etc.), Nonpolar side chains (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, etc.), β-branched side chains (threonine, valine, isoleucine, etc.) and Aromatic side chains (tyrosine, phenylalanine, tryptophan, histidine, etc.) The amino acids include those having the formula:

[0040] Other conservative amino acid substitutions can also occur across amino acid side chain families, such as substituting aspartic acid for asparagine to modify the charge of the peptide. Conservative changes can also include the substitution of chemically homologous non-natural amino acids (i.e., a synthetic non-natural hydrophobic amino acid for leucine, a synthetic non-natural aromatic amino acid for tryptophan).

[0041] According to one embodiment of the invention, the human CD70b to which the antibody or fragment binds is a) an amino acid sequence set forth in any one of SEQ ID NOs: 41 to 44, or b) an amino acid sequence having at least 80% sequence identity with SEQ ID NOs: 41 to 44, but maintaining CD79b activity; Includes:

[0042] In some embodiments, human CD79b comprises an amino acid sequence having 81% or more, preferably 82% or more, more preferably 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or most preferably 99% or more sequence identity to SEQ ID NOs: 41-44.

[0043] SEQ ID NO: 41 represents the amino acid sequence of human CD79b and is accessible at the NCBI reference. Generally, various variants and isoforms of CD79b exist, and are disclosed herein as SEQ ID NOs: 42-44. Similarly, variants containing conservative or silent amino acid substitutions that retain full or at least substantial CD79b activity exist or may exist. These isoforms, variants, and mutants are encompassed within the identity ranges defined above, but functionally inactive variants and mutants are excluded.

[0044] In this regard, the present inventors have surprisingly found that the antibodies according to the present invention bind to all four isoforms of CD79b mentioned above, since existing anti-CD79b antibodies such as SN8 recognize the epitope ARSEDRYRNPKGSACSRIWQS (SEQ ID NO: 61), which is present only in isoforms 1 and 3, but not in isoforms 2 and 4 (see Figure 9 for an alignment of the different isotypes). Moreover, the antibodies according to the present invention have a surprisingly broad target spectrum.

[0045] According to one embodiment of the present invention, the antibody or fragment is a monoclonal antibody, or a target-binding fragment or derivative thereof, that retains target-binding ability to human CD79b.

[0046] According to one embodiment of the present invention, the antibody or fragment is in at least one format selected from the group consisting of: IgG, scFv, Fab, or (Fab)2.

[0047] As used herein, the term "monoclonal antibody (mAb)" refers to an antibody composition having a homogeneous antibody population, i.e., a homogeneous population of whole immunoglobulins or fragments or derivatives thereof that retain target binding ability.

[0048] Particularly preferably, such antibodies are IgG antibodies, or fragments or derivatives thereof that retain target binding ability. Immunoglobulin G (IgG) is a type of antibody. IgG accounts for approximately 75% of human serum antibodies and is the most common type of antibody present in the blood. IgG molecules are produced and released by plasma B cells. Each IgG has two antigen-binding sites.

[0049] IgG antibodies are large molecules with a molecular weight of approximately 150 kDa, consisting of four peptide chains. They contain two identical class γ heavy chains of approximately 50 kDa and two identical light chains of approximately 25 kDa, resulting in a tetrameric structure. The two heavy chains are linked to each other and to the light chains by disulfide bonds. As a result, the tetramer has two identical halves that together form a Y-shape. Both ends of the fork contain identical antigen-binding sites. The Fc region of IgG contains a highly conserved N-glycosylation site. N-glycans bound to this site are primarily complex-type, core-fucosylated diantennary structures. Furthermore, a small proportion of these N-glycans have been observed to contain a bisecting GlcNAc and α-2,6-linked sialic acid residues.

[0050] According to one embodiment of the present invention, the antibody is in at least one format selected from the group consisting of IgG1, IgG2 or IgG4.

[0051] As used herein, the term "fragment" refers to fragments of such antibodies that retain target binding ability. For example, CDR (complementarity determining region) hypervariable region, Variable domain (Fv) IgG or IgM heavy chain (consisting of VH, CH1, hinge, CH2 and CH3 regions) an IgG or IgM light chain (consisting of a VL and CL region), and / or Fab and / or F(ab)2.

[0052] As used herein, the term "derivative" refers to protein constructs that are structurally different but still have some structural relatedness, such as scFv, Fab and / or F(ab)2, as well as dual, tri- or higher specificity antibody constructs, and the common antibody concept, which still retain target binding ability, all of which are described below.

[0053] Other antibody derivatives known to those skilled in the art are diabodies, camelid antibodies, nanobodies, domain antibodies, bivalent homodimers with two chains consisting of scFvs, IgAs (two IgG structures linked by a J chain and a secretory component), shark antibodies, antibodies consisting of a New World primate framework and non-New World primate CDRs, dimerization constructs containing CH3+VL+VH, and antibody conjugates (e.g., antibodies or fragments or derivatives linked to toxins, cytokines, radioisotopes, or labels). These types are well described in the literature and can be used by those skilled in the art based on the present disclosure without further inventive effort.

[0054] Methods for producing hybridoma cells are disclosed in Kohler and Milstein (1975).

[0055] Methods for the production and / or selection of fully human mAbs are known in the art and include the use of transgenic animals immunized with the respective protein or peptide, or the use of suitable display technologies such as yeast display, phage display, B cell display, or ribosome display, where antibodies from a library are screened against human CD79b in stationary phase.

[0056] In vitro antibody libraries are disclosed, inter alia, by MorphoSys in US6300064 and by MRC / Scripps / Stratagene in US6248516. Phage display technology is disclosed, for example, by Dyax in US5223409. Transgenic mammalian platforms are described, for example, in EP1480515A2 by TaconicArtemis.

[0057] IgG, IgM, scFv, Fab and / or F(ab)2 are antibody formats well known to those skilled in the art. The relevant validation techniques are available from the respective textbooks.

[0058] As used herein, the term "Fab" refers to an IgG / IgM fragment containing the antigen-binding region, said fragment consisting of one constant and one variable domain from each of the heavy and light chains of the antibody.

[0059] As used herein, the term "F(ab)2" refers to an IgG / IgM fragment consisting of two Fab fragments linked together by disulfide bonds.

[0060] As used herein, the term "scFv" refers to a single-chain variable fragment that is a fusion of the variable regions of the heavy and light chains of an immunoglobulin, connected by a short linker, usually serine (S) or glycine (G). This chimeric molecule retains the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of the linker peptide.

[0061] Modified antibody formats are, for example, bi- or trispecific antibody constructs, antibody-based fusion proteins, immunoconjugates, etc. These types are well described in the literature and can be used by the skilled artisan based on the present disclosure to further add activity to the invention.

[0062] In one or more embodiments, the antibody or fragment thereof is an isolated antibody, or a target-binding fragment or derivative thereof that retains target binding ability, or an isolated antibody mimetic. In one or more embodiments, the antibody is an engineered or recombinant antibody, or a target-binding fragment or derivative thereof that retains target binding ability, or an engineered or recombinant antibody mimetic.

[0063] According to one embodiment of the present invention, the antibody or fragment is an antibody in at least one format selected from the group consisting of: IgG, scFv, Fab, or (Fab)2.

[0064] According to another aspect of the invention there is provided a nucleic acid encoding at least one strand of a binding agent according to the above description.

[0065] In one embodiment, when the binding agent is a monoclonal antibody having a heteromeric structure of at least one light chain and one heavy chain, a nucleic acid or a pair of nucleic acids encoding the heavy and light chains of the binding agent, respectively, is provided.

[0066] Such nucleic acids can be used for the recombinant production of the antibody or a fragment or derivative thereof in a suitable expression system, such as, for example, CHO cells or Nicotinic acid.

[0067] Such nucleic acids can also be used for pharmaceutical purposes. The nucleic acid can be an RNA molecule or an RNA derivative containing modified nucleotides such as pseudouridine (Ψ) or N-1 methylpseudouridine (mΨ) to provide stability and reduce immunogenicity (see, e.g., US8278036 and US9428535, the contents of which are incorporated herein by reference for purposes of enablement). In another embodiment, RNA containing the most GC-rich codons is selected to provide stability and reduce immunogenicity (see, e.g., EP1392341, the contents of which are incorporated herein by reference for purposes of enablement). The mRNA can be delivered, for example, in appropriate liposomes, and contains either specific sequences or modified uridine nucleosides to avoid immune responses and / or improve folding and translation efficiency, and sometimes contains cap modifications at the 5' and / or 3' ends to target them to specific cell types.

[0068] The nucleic acid may also be a DNA molecule. In this case, the molecule may be cDNA, optionally incorporated into a suitable vector, such as an attenuated, non-pathogenic virus, or provided as one or more plasmids. Such a plasmid may be administered to a patient using an electroporation device, such as that disclosed in patent EP3397337B1 (the contents of which are incorporated herein by reference for purposes of enablement).

[0069] Generally, due to the degeneracy of the genetic code, there are many nucleic acids capable of encoding such a chain. A person skilled in the art is fully capable of determining whether a given nucleic acid satisfies the above criteria. On the other hand, a person skilled in the art is perfectly capable of reverse engineering a suitable nucleic acid encoding a given amino acid sequence based on a codon usage table. For this purpose, software tools such as "reverse translate" provided by the online tool "sequence manipulation suite" (https: / / www.bioinformatics.org / sms2 / rev_trans.html) can be used. Therefore, there are numerous alternative DNA and RNA sequences that code for the protein sequence described in the claims. These alternative sequences are considered to be within the scope of the present invention.

[0070] According to another aspect of the present invention there is provided a recombinant immunotoxin, immunocytokine, antibody drug conjugate or antibody-radionuclide conjugate, which embodiment comprises an antibody or fragment according to the above description.

[0071] As used herein, the term "recombinant immunotoxin" refers to a fusion construct comprising at least (i) one antibody or fragment according to the above description and (ii) a protein toxin or protoxin fused thereto. Such recombinant immunotoxins can be produced in a suitable recombinant expression system without the need for subsequent conjugation of the toxin to the antibody or fragment.

[0072] As used herein, the term "protein toxin" or "protein protoxin" is meant to encompass cytotoxic and / or cytostatic proteins, or pro-variants thereof.

[0073] As used herein, the term "cytostatic protein" refers to a protein that can inhibit cell growth or cell division without necessarily killing the cell. Suitably, a cytostatic agent inhibits the growth of tumor cells.

[0074] As used herein, the term "cytotoxic protein" refers to a protein that is harmful to cells and ultimately causes cell death. In some embodiments, a cytotoxic protein damages rapidly dividing cells, such as tumor cells, causing tumor cell death, particularly tumor cell death, while causing no or less damage to non-tumor cells.

[0075] The term "protein toxin" or "protein protoxin" does not exclusively refer to toxins that are, by their chemical nature, proteins (i.e., peptides having a length of 50 amino acid residues or more) or polypeptides (i.e., peptides having a length of 10 to 50 amino acid residues or more). Protoxins in the sense of the present invention are precursors of toxins, also called latent toxins, which must be activated, for example, by cleaving an inhibitory amino acid sequence or by undergoing a conformational change. The terms "protoxin" and "protein protoxin" are used interchangeably herein and refer to the same entity.

[0076] Such protein toxins or protoxins may be selected from the group consisting of ribotoxins, endoribonucleases (RNases), ribosome-inactivating proteins (RIPs) and type AB toxins.

[0077] The term "ribotoxin" as used herein refers to a group of extracellular ribonucleases secreted by fungi. Their most notable feature is their specificity. Ribosomes are inactivated by cleaving a single phosphodiester bond in rRNA at a universally conserved sequence. This cleavage triggers cell death by apoptosis. However, as extracellular proteins, they must first enter the target cell to exert their cytotoxic effect. This entry constitutes a rhythmic step in their activity.

[0078] Ribotoxins have been detected in a variety of fungi, including entomopathogenic and edible species. However, the three-dimensional structures of only three have been elucidated: α-sarcin (SEQ ID NO: 56 and its deimmunized variant SEQ ID NO: 57), restrictocin, and hirsuterin A (HtA, SEQ ID NO: 48). The first two are produced by Aspergillus giganteus and Aspergillus restrictus, respectively, and are nearly identical. HtA, produced by the entomopathogenic fungus Hirsutella thompsonii, is much smaller, sharing only 25% sequence identity with other large ribotoxins. Nevertheless, it retains all of the functional characteristics of the family. A second ribotoxin similar to HtA, anisopurine (SEQ ID NO: 49, and its analogs and deimmunized variants, disclosed herein as SEQ ID NOs: 50, 52, 53, and 54), is known (70% sequence identity with HtA). It is produced by the fungus Metarhizium anisopliae, which is also an insect pathogen. Other ribotoxins that can be used in the context of the present invention are angiogenin (SEQ ID NO: 51) and ageritin (SEQ ID NO: 55).

[0079] The term "RNase" as used herein relates to a group of catalytic nucleases ("ribonucleases") that degrade RNA into smaller components. In the sense of the present invention, ribonucleases act as endoribonucleases. In some embodiments, the RNase is one selected from the following group: Onconase: (Rampirinase, Frog RNase): Different variants of onconase exist, examples of which are published under the UniProt identifiers Q8UVX5, Q9I8V8, Q6EUW9, Q6EUW8, Q6EUW7 or P22069. Some examples in this application use Q8UVX5, however other onconase variants can be used as well.

[0080] RNase 1: pancreatic ribonuclease (e.g., hRNase 1, e.g., Uniprot identifier P07998 (SEQ ID NO: 58) RNase 5: angiogenin (e.g., hRNase 5, e.g., Uniprot identifier P03950) RNase 2: non-secretory ribonuclease (e.g., hRNAse 2, e.g., Uniprot identifier P10153) RNase 3: eosinophil cationic protein (e.g., hRNAse 3 / Drosha, e.g., Uniprot identifier Q9NRR4 or P12724) RNase 4: Ribonuclease 4 (e.g., hRNAse 4, e.g., Uniprot identifier P34096) RNase 6: ribonuclease K6 / ribonuclease T2 / ribonuclease K3 (e.g., hRNAse6, e.g., Uniprot identifier Q93091) RNase 7: Ribonuclease 7 / Ribonuclease A E1 (e.g., RNAse7, e.g., Uniprot identifier Q9H1E1) RNase 8: Ribonuclease 8 (e.g., hRNAse 8, e.g., Uniprot identifier Q8TDE3) The above Uniprot identifiers are for illustrative purposes only. Other variants may also be used. Those skilled in the art can find such variants in the respective databases with routine effort.

[0081] Ribosome-inactivating proteins (RIPs) are toxic N-glycosidases that depurinate rRNA in eukaryotes and prokaryotes, thereby halting protein synthesis during translation. RIPs are widely distributed in various plant species and tissues. These proteins are known to play an important role in defense against pathogens and have been suggested to confer disease resistance. Plant-derived RIPs have been identified in over 50 species from 14 families, including Cucurbitaceae, Euphorbiaceae, Poaceae, and Caryophyllaceae. RIPs have also been found in bacteria, fungi, algae, and even mosquitoes.

[0082] RIPs constitute a large family of proteins that can be classified according to their structural composition into RIP type I and type II.

[0083] Type I RIPs are small proteins with a molecular weight of around 30 kD, and occur as single-chain proteins. The single chain of type I RIPs consists of an enzymatically active domain (A domain or α domain) that exerts N-glycosidase activity.

[0084] Type II RIPs are large proteins of 50-65 kDa, characterized by an enzymatically active A chain and a slightly larger B chain (or β chain, lectin subunit) bearing a galactose-like sugar chain.

[0085] In addition to RIP types I and II, a third class, type III, has been described, with a small number of members containing an N-terminal domain that correlates with the A domain of RIPs, fused to a C-terminal domain of unknown function.

[0086] According to some embodiments, the ribosome-inactivating protein (RIP) is at least one selected from the group consisting of: Momordin, Bryodin I (SEQ ID NO: 62) Kukurumosin, Bryodin II (SEQ ID NO: 63) Trichosanthin, Caraslin, MOMC, ME1, and / or ME2.

[0087] Type AB toxins are two-component protein complexes secreted by many pathogenic bacteria. These toxins are classified as type III toxins because they disrupt intracellular functions. They are named for their components: the "A" component is usually the "active" part, and the "B" component is usually the "binding" part. The "A" subunit possesses enzymatic activity and is translocated into host cells through a conformational change in the membrane-bound transport "B" subunit. These proteins consist of two separate polypeptides corresponding to the A / B subunit moieties. The enzymatic component (A) enters the cell through an endosome produced by the oligomer-binding / translocation protein (B) and inhibits actin polymerization by ADP-ribosylation of monomeric G-actin.

[0088] Examples of the "A" component of type AB toxins include C. perfringens iota toxin Ia, C. botulinum C2 toxin CI, and Clostridium difficile ADP-ribosyltransferase. Other homologous proteins have been found in Clostridium spiroformes.

[0089] An example of the B component of a type AB toxin is the protective antigen (PA) protein of Bacillus anthracis, which secretes three toxin factors (protective antigen (PA), edema factor (EF), and lethal factor (LF)), each of which is a heat-degradable protein of ~80 kDa. PA forms the "B" portion of the exotoxin, allowing the "A" portion (consisting of EF or LF) to enter the target cell. The PA protein forms the central part of the complete anthrax toxin, which assembles as a heptamer in the membrane before translocating the A portion into the host cell.

[0090] Diphtheria toxin is also a type AB toxin. It inhibits host cell protein synthesis by phosphorylating eukaryotic elongation factor 2, which is essential for protein synthesis. Pseudomonas aeruginosa exotoxin A is another example of a type AB toxin that targets eukaryotic elongation factor 2.

[0091] AB5 toxins are usually considered a subtype of AB toxins, characterized by the B pentamer. Less commonly, the term "AB toxins" is used to emphasize the monomeric nature of the B component.

[0092] The biphasic mechanism of action of AB toxins has attracted particular attention in cancer therapy research. The general idea is to modify the B component of an existing toxin so that it binds selectively to malignant cells. This approach combines the benefits of cancer immunotherapy with the high toxicity of AB toxins, creating a new class of chimeric protein drugs called immunotoxins.

[0093] In one embodiment, the recombinant immunotoxin comprises: i) an antibody according to the above description; ii) Toxins as described above and iii) a peptide linker connecting the two elements i) and ii). Includes.

[0094] Suitable linkers for this purpose are disclosed herein as SEQ ID NOs: 45, 46, 47 and 60.

[0095] In one embodiment, the recombinant immunotoxin comprises antibody 580 defined by the amino acid sequence of SEQ ID NOs: 3-10 (SEQ ID NOs: 3 and 4: variable domains, SEQ ID NOs: 5-10: CDRs).

[0096] In one embodiment, the recombinant immunotoxin comprises antibody 704 comprising the amino acid sequence of SEQ ID NOs: 33-40 (SEQ ID NOs: 33 and 34: variable domain, SEQ ID NOs: 35-40: CDR). In one embodiment, the recombinant immunotoxin comprises the ribotoxin anisopurine or an analog (selected from any one of SEQ ID NOs: 49, 50, 52-54). In one embodiment, the recombinant immunotoxin comprises a peptide linker that is not cleaved by mammalian proteases, such as a G4S linker (SEQ ID NO: 60).

[0097] In one embodiment, the recombinant immunotoxin comprises: (i) antibody 580 defined by the amino acid sequences of SEQ ID NOs: 3 to 10; (ii) the ribotoxin anisoplin having the sequence of SEQ ID NO: 49, and (iii) a peptide linker having the sequence of SEQ ID NO: 60 Includes.

[0098] In one embodiment, the recombinant immunotoxin comprises: (i) antibody 704, defined by the amino acid sequence of SEQ ID NOs: 33 to 40; (ii) the ribotoxin anisoplin having the sequence of SEQ ID NO: 49, and (iii) a peptide linker having the sequence of SEQ ID NO: 60 Includes. Preferably, the antibodies in the recombinant immunotoxin are of the IgG1 format.

[0099] As used herein, the term "immunocytokine" refers to a fusion construct comprising at least (i) one antibody or fragment according to the above description and (ii) an immunomodulatory cytokine fused thereto. Such recombinant immunocytokine can be produced in a suitable recombinant expression system without the need for subsequent conjugation of the cytokine to the antibody or fragment.

[0100] Immunocytokines can be used to improve site-specific delivery and extend the half-life of cytokines. Although immunocytokines are administered systemically, they can be specifically targeted via specific tumor antigens. Cytokines suitable for fusion to antibodies include TNFα, IL2, IL12, and IL15. In this way, the maximum tolerated dose can be increased, and for IL12, it was found to be 30-fold higher than that of IL12 alone.

[0101] As used herein, the term "antibody drug conjugate" refers to a construct comprising an antibody or fragment thereof to which a toxin is covalently attached. The toxin is typically a small molecule toxin having a molecular weight of 2500 Da or less, and is often selected from the group consisting of: Maytansines Monomethyl auristatin Calcare sewing machine, Doxorubicin pyrrolobenzodiazepines, methotrexate, Topoisomerase 1 inhibitors Glucocorticoid receptor modulators (GRMs) Taxanes Anthracyclines α-amanitin, and / or Cyclosporines Some examples include, but are not limited to, SN38, exatecan, dexamethasone, budesonide, mertansine, ansamitocin, rabatasine, DM4, DM1, ozogamicin, monomethyl auristatin F (MMAF) monomethyl auristatin E (MMAE).

[0102] In contrast to immunotoxins and immunocytokines, effectors must be conjugated to antibodies in a separate step, typically by using a linker / spacer containing a para-aminobenzyl carbamate attached to the free thiol group of a cysteine ​​residue in the antibody, a cathepsin-cleavable linker containing citrulline and valine, or a linker consisting of a caproic acid and maleimide group, followed by attachment of the toxin.

[0103] Here, issues of site specificity of the conjugation reaction and stochasticity between the antibody and the toxin play a key role. One attempt to solve such problems involves the so-called thiomab approach, in which antibodies are cysteine ​​engineered to create preferred conjugation sites for linkers / spacers (see Panowski et al., 2014).

[0104] Other attempts utilize specific enzymes, such as sortase or transglutaminase, that can be used to conjugate toxins to antibodies in a stoichiometric and site-specific manner (see, e.g., WO2014140317A1 and WO2020188061A1).

[0105] Antibody-Radionuclide is Yttrium 90 , iodine 131 ,lutetium 177 Such molecules include antibodies or fragments thereof labeled with at least one radionuclide, such as, for example, those disclosed in Steiner & Neri 2011, the contents of which are incorporated herein by reference for purposes of enabling.

[0106] According to another aspect of the present invention, there is provided a pharmaceutical composition comprising an antibody or fragment as described above, a nucleic acid as described above, or a recombinant immunotoxin, immunocytokine, antibody drug conjugate, or antibody-radionuclide as described above, and optionally one or more pharmaceutically acceptable excipients.

[0107] According to another aspect of the present invention, there is provided a combination comprising (i) an antibody or fragment as described above, a nucleic acid as described above, a recombinant immunotoxin, an immunocytokine, an antibody drug conjugate or an antibody-radionuclide as described above, or a pharmaceutical composition as described above, and (ii) one or more therapeutically active compounds.

[0108] According to another aspect of the present invention there is provided the use of an antibody or fragment as described above, a nucleic acid as described above, a recombinant immunotoxin, an immunocytokine, an antibody drug conjugate or an antibody-radionuclide as described above, a pharmaceutical composition as described above or a combination as described above for the treatment of a human or animal subject diagnosed with, suffering from or at risk of developing a neoplastic disease, or for the prevention of such a condition (for the manufacture of a medicament).

[0109] This wording is deemed to encompass both the Swiss-type claim wording accepted in some countries (in which case the brackets are deemed to be absent) and the EPC 2000 wording (in which case the brackets and any content within them are deemed to be absent).

[0110] According to another aspect of the present invention there is provided a method for treating or preventing a neoplastic disease, the method comprising administering to a human or animal subject a therapeutically sufficient dose of an antibody or fragment as described above, a nucleic acid as described above, a recombinant immunotoxin, an immunocytokine, an antibody drug conjugate or an antibody-radionuclide as described above, a pharmaceutical composition as described above or a combination as described above.

[0111] According to another aspect of the present invention, there is provided a kit for the treatment of a portion, comprising: a) an antibody or fragment as described above, a nucleic acid as described above, a recombinant immunotoxin, an immunocytokine, an antibody drug conjugate or an antibody-radionuclide as described above, a pharmaceutical composition as described above, or a combination as described above; a composition, a device for administering the composition or combination, and c) Instructions for use. [Example]

[0112] While the invention has been illustrated and described in detail in the drawings and the foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive, and the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0113] All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5'->3'.

[0114] Materials and Methods Genetic construct of anti-CD79B antibody The full-length rituximab HC and LC sequences have been used to develop mAb-based binder-toxin fusion proteins. We developed the heavy and light chain variable subsequences of anti-CD79B from humanized murine antibody SN8 and fused them to a human IgG1 Fc subsequence.

[0115] Gene construct containing anti-CD79B antibody An anti-CD79B antibody was developed using a hybrid humanization strategy of a murine antibody (SN8). The full-length HC and LC antibody sequences were used to develop antibody-based binder-toxin fusion proteins. An anisopurine sequence was then fused to the C-terminal portion of the HC using a G4S sequence, respectively, to yield HC-G4S-anisopurine + LC. Other binder-toxin fusion proteins were constructed by combining the scFv-Fc, HC, and LC portions with anisopurine or other ribotoxins containing cleavage sites, yielding HC-FCS-anisopurine + LC, HC-FCS-anisopurine + LC, and LC-FCS-anisopurine + HC-FCS-anisopurine. These sequences were generated by gene synthesis flanked by XbaI and IsceI.

[0116] Transient expression in Nicotiana benthamiana leaves Nicotiana benthaminana was grown under a 16-hour light / 8-hour dark photocycle at 22±3°C. Leaves from 7-8 week-old plants were transiently transformed by syringe infiltration. The optical density at 600 nm (OD 600 Agrobacterium tumefaciens GV3101 (pMP90) carrying an unpublished plasmid containing a gene construct with an OD of 0.8–1.0 was harvested by centrifugation at 3500 g for 10 min. Finally, the bacteria were grown in infiltration buffer (10 mM MgCl2, 10 mM MES, 100 μM acetosyringone, pH 5.6) until the OD reached 0.8–1.0. 600 The pH was adjusted to 0.5, and the leaves were infiltrated using a needleless syringe. The infiltrated areas were harvested on days 4 and 6 after agroinfiltration. Whole leaves harvested on day 4 after agroinfiltration were used for protein A purification.

[0117] Expression in N. tabacum cells Nicotiana tabacum plant suspension cells were cultured in the plant culture medium described by Nagata et al. (1992), the contents of which are incorporated herein, at 130 rpm and 25°C for 5 days. The optical density at 600 nm (OD 600Agrobacterium tumefaciens LBA4404 (pBBR1MCS-5.virGN54D) harboring the pPZP-ATB binary plasmid, with a chromatin density of 0.8–1.0, was harvested by centrifugation at 2000 g for 5 minutes. Plant and bacterial cells were then co-cultured in co-culture medium for 30 minutes, followed by centrifugation at 2000 g for 5 minutes. After removing the supernatant, the cells were plated on solid co-culture medium for two days. For transient transformation, the cells were then harvested, washed three times, and cultured on plant growth medium containing cefotaxime and carbenicillin before being harvested for further analysis. For stable transformation, after two days of solid co-culture, the cells were washed and plated on plant medium containing selective antibiotics kanamycin, cefotaxime, and carbenicillin. After four weeks, calli were selected and subcultured on solid medium or in liquid suspension for further analysis.

[0118] Protein A purification Four days after agroinfiltration, leaves were collected, weighed, and ground in a blender with 2 mL of extraction buffer (0.1 M TRIS, 460 mM NaCl, 5 mM EDTA, 5 mM sodium metabisulfite, pH 7.5) per gram of freshly agroinfiltrated leaves. The mixture was then filtered through a double Miracloth (Millipore) layer. The filtrate was centrifuged at 40,000 xg for 10 minutes at 4°C. The supernatant was then loaded onto Protein A resin pre-equilibrated with wash buffer. The resin was then washed with 10 column volumes of 60 mM TRIS, 25 mM, 460 mM NaCl, pH 7.5, and eluted with 100 mM glycine, 460 mM NaCl, pH 3.0, directly buffered with 10% Tris, 1 M, pH 8.0. The concentrated protein fraction was collected, dialyzed, and frozen in liquid nitrogen. The purified binder-toxin proteins were visualized by SDS-PAGE.

[0119] Size exclusion chromatography After Protein A, the concentrated protein fraction is loaded onto a Sephacryl S-300 HR column (Cytivia). The main pick is collected and residual pick is removed from the collected fractions before pooling.

[0120] Protein analysis: ELISA, SDS-PAGE and Western blot Proteins were boiled in reducing or non-reducing SDS loading buffer (80 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.005% bromophenol blue) for 5 min, centrifuged at 13,000 rpm for 5 min, and separated by SDS-PAGE (4-20% polyacrylamide).

[0121] For Western blotting, proteins were electrophoresed onto a PVDF membrane (Biorad) using a semi-dry electrophoresis apparatus (Biorad Trans-Blot Turbo); the membrane was then blocked with 3% (w / v) nonfat dry milk in TBST buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% Tween 20, pH 7.5) for 1 h at room temperature and then incubated (TBS-Tween 0.1% + 0.5% nonfat dry milk) for 1 h at room temperature with a 1:10,000 dilution of anti-human IgG Fc-specific region (A0170; Sigma-Aldrich) or a polyclonal antibody against anisopurine (internal reference) at a 1:50,000 dilution. After the anti-human Fc and anti-anisopurine antibodies, an HRP-conjugated anti-rabbit antibody (Synabs) was added at a 1:5,000 dilution. Proteins were detected by enhanced chemiluminescence (Amersham Imager 600 / GE; GE Healthcare).

[0122] Protein Analysis Size Exclusion Chromatography The purified protein was analyzed by size-exclusion chromatography using a Cytiva high-resolution Superdex 200 Increase 10 / 300 GL column on an Äkta Pure or Äkta Go chromatography system. The column was equilibrated with two volumes of PBS at a flow rate of 1 mL / min. Purified protein samples of 1–10 mg / mL were centrifuged at 20,000 g for 5 minutes. The supernatant was then collected, and 200 μL was loaded into a 100 μL capillary sample loop. The sample in the loop was then loaded onto the column at a flow rate of 0.75 mL / min and eluted with 1.5 CV of PBS. The elution profile was monitored by absorbance (UV).

[0123] In vitro cytotoxicity test The effect of binder-toxin fusion proteins on the viability of CD79b-expressing cell lines was assessed using the Cell Titer Glo Assay (Promega, G9241). 2+ In the presence of ATP, luciferase catalyzes the mono-oxygenation of luciferin, which generates a luminescent signal proportional to the number of viable cells.

[0124] Depending on the cell line tested, cells were seeded into wells of a 96-well plate at a density of 2,000 or 5,000 cells / well in 50 μl of growth medium (RPMI 1640). Serial dilutions of binder-toxin fusions were prepared by adding 10 μl of binder-toxin fusion or buffer (PBS, Tween 0.02%) to 40 μl of growth medium. The mixtures were added to the cells and incubated at 37°C and 5% CO2 for 72 hours. Binder-toxin fusions were tested in duplicate. Buffer was used as a negative control, and medium and cells alone served as blank and untreated controls, respectively.

[0125] After 72 hours, the plate was equilibrated at room temperature for 30 minutes, and 100 μl of CellTiter Glo reagent was added to each well. The plate was then placed on a shaking platform for 2 minutes, and the signal was allowed to stabilize for 10 minutes in the dark at room temperature. Luminescence was then recorded.

[0126] Viability was determined by subtracting the mean luminescence signal from the blank (growth medium only) from each well, and the mean luminescence signal from untreated cells was set as 100% viability. The mean signal from treated cells was then normalized and plotted as a function of binder-toxin fusion concentration.

[0127] Anti-CD79b-based binder-toxin fusion proteins were evaluated in B-cell lymphoma cell lines (B-NHL, CD79+) and the non-target cell line K562 (CD79-).

[0128] Immunohistochemistry - Tissue Microarray (TMA) The binder-toxin fusion was labeled with fluorescein isothiocyanate (FITC) using Thermo Scientific's antibody labeling kit "Pierce."

[0129] Slides containing CD79b-positive cell lines (representative of B-NHL) and CD79b-negative cells (K562) were used to define the optimal antibody concentration for method development and further studies. For cross-reactivity testing, TMAs containing human (T6234701-1 / 2) normal frozen tissues were used (Biochain Institute Incorp, CA USA).

[0130] Fixation was performed in cold acetone for 10 minutes, followed by 10 minutes of air drying. PBS + 10% normal human serum (Jackson Immunoresearch, 009-000-121) was added for 20 minutes. Normal human serum was removed, and tissue blocks were incubated with FITC-labeled binder-toxin fusion for 1 hour. Blocks were washed with PBS for 3 minutes, followed by incubation with rabbit anti-FITC (Serotec, #4510-7804) at a 1:1000 dilution for 30 minutes.

[0131] The material is washed for 3 minutes in PBS and then incubated for 30 minutes in ready-to-use polymer BrightVision anti-rabbit / HRP (Immunologics, DPVR110HRP). The tissue block is washed for 3 minutes in TRIS buffer (0.05M Trizmabase - Sigma, T1503-500g - in distilled water). The blocked tissue is incubated for 5 minutes in DAB (50mg of diaminobenzidine - Sigma, #D5637-5G - dissolved in 100ml of Tris buffer (see above) and 100ml of H2O2 - Merck, 1.07209.0250 - added immediately before incubation).

[0132] Wash the tissue blocks in distilled water for 3 minutes, counterstain with hematoxylin for 10 seconds, and then quickly wash them in distilled water. Dehydrate the tissue blocks in 70, 90, and 95% ethanol for 2 minutes each, then in 99.5% ethanol for 5 minutes twice, and in X-trasolve (Medite, 41-5213-00) for 5 minutes three times. Prepare sections with coverslips and mounting media (Medite, 41-5219-00).

[0133] Staining was judged as negative (0), weak (1+), weak to moderate (1-2+), moderate (2+), moderate to strong (2-3+), or strong (3+).

[0134] Cell line-derived xenografts Representative B-NHL cell lines were injected into the peritoneum of CB17.SCID mice. Tumors were 1 cm 3 Once tumors reached a desired size, CD79B expression was confirmed by FACS analysis, and the tumors were cut into small pieces and implanted into CB17.SCID mice. Once the average tumor volume reached the desired size, animals were transplanted to a mean tumor volume of 0.139 cm. 3 The animals were stratified into 6 groups of 7 each.

[0135] All binder-toxin fusions used in vivo were aggregate-free and purified by SEC. Animals were intravenously administered a single 20 mg / kg dose of ATB-747, ATB-580, ATB-693, ATB-697, and ATB-704 or vehicle (PBS, tween 0.02%). Body weight and tumor volume were monitored three times weekly.

[0136] The mean tumor volume for each group was plotted against the number of days after treatment initiation. Tumor growth curves were plotted for individual animals in each group.

[0137] For ethical reasons, tumor volumes of 1.5 cm 3 Mice were sacrificed when the time approached 100°C.

[0138] Statistical analysis was performed using GraphPad Prism 9.3.1. Two-way analysis of variance was performed to compare different groups.

[0139] Cell viability measurement method The purified binder-toxin fusions were evaluated for cytotoxicity in cancer cell lines. All humanized anti-CD79B toxin fusions (ATB-580-693-697-704) were shown to impair the viability of CD79B-positive cell lines. Furthermore, they demonstrated superiority over Polivy (polatuzumab vedotin), a commercially available ADC targeting CD79B. These results demonstrate the superior mode of action of binder-toxin fusions compared to antibody-drug conjugates.

[0140] Off-target / cross-reactivity assessment Figure 7 shows that all humanized anti-CD79B toxin fusions (ATB-580-693-697-704) showed no off-target binding. Only tissues containing targeted B cells were stained (lymph nodes, spleen, and thymus). Similar data were obtained with frozen tissues from cynomolgus monkeys and mice (data not shown).

[0141] In vivo efficacy Polatuzumab As shown in Figure 8, all humanized recombinant anti-CD79B immunotoxins (ATB-580-693-697-704) demonstrated potent and long-lasting antitumor efficacy in the CDX model. In particular, ATB-580 outperformed the benchmark polatuzumab recombinant immunotoxin ATB-747 (fused to the protein toxin anisopurine via a G4S linker) (Figure 8) (polatuzumab).

[0142] Size Exclusion Chromatography (SEC) The purified protein was analyzed by size-exclusion chromatography using a Cytiva high-resolution Superdex 200 Increase 10 / 300 GL column on an Äkta Pure or Äkta Go chromatography system. The column was equilibrated with two volumes of PBS at a flow rate of 1 mL / min. Purified protein samples at 1–10 mg / mL were centrifuged at 20,000 g for 5 minutes. The supernatant was then collected, and 200 μL was loaded into a 100 μL capillary sample loop. The sample in the loop was then loaded onto the column at a flow rate of 0.75 mL / min and eluted with 1.5 CV of PBS. The elution profile was monitored by absorbance (UV).

[0143] Analytical SEC demonstrated low aggregation propensity for humanized anti-CD79B toxin fusions, ranging from 3% (ATB-704) to 8% (ATB-508) of aggregates (Figure 4). Humanized anti-CD79B toxin fusions were observed to have significantly lower aggregation propensity compared to the benchmark, polatuzumab (ATB-452), which exhibited 13% aggregates (Figure 5).

[0144] References [Table A]

[0145] array The following sequences form part of the disclosure of this application. A WIPO ST 26-compatible electronic sequence listing is also provided with this application. For the avoidance of doubt, in the event of any discrepancy between a sequence in the table below and a sequence in the electronic sequence listing, the sequence in the table shall be deemed correct. In some cases, a signal peptide may be included in the reproduced sequence. In this case, the sequence shall be deemed to be disclosed with or without the signal peptide. A readily available tool for identifying signal peptides in a given protein sequence is SignalP - 6.0, provided by Dansk Technical University under https: / / services.healthtech.dtu.dk / service.php?SignalP

[0146] Also, note that in some embodiments, the amino acid sequence of each of the toxins represents a deimmunized version thereof, and all embodiments are considered to be disclosed using either the wild-type toxin sequence or the deimmunized variant.

[0147] array [Table 4] JPEG2025530642000007.jpg248170 JPEG2025530642000008.jpg249170 JPEG2025530642000009.jpg244170 JPEG2025530642000010.jpg238170

Claims

1. An antibody or target-binding fragment or derivative thereof that retains target binding ability to human CD79b, a) SEQ ID NOs: 3 and 4; SEQ ID NOs: 13 and 14, SEQ ID NOs: 23 and 24, or SEQ ID NOs: 33 and 34 or a set of six heavy / light chain complementarity determining regions (CDRs) contained in a heavy chain / light variable domain sequence pair selected from one of the pairs b) in the order of HCDR1; HCDR2; HCDR3; LCDR1; LCDR2 and LCDR3, i) SEQ ID NOs: 5, 6, 7, 8, 9, and 10; ii) SEQ ID NOs: 15, 16, 17, 18, 19, and 20; iii) SEQ ID NOs: 25, 26, 27, 28, 29, and 30, or iv) SEQ ID NOs: 35, 36, 37, 38, 39, and 40 or a set of six heavy / light chain complementarity determining regions (CDRs) selected from c) a set of heavy / light chain complementarity determining regions (CDRs) according to option b), with the proviso that at least one of the CDRs has up to three amino acid substitutions relative to the CDR contained in the respective SEQ ID NO:; and / or d) a set of heavy / light chain complementarity determining regions (CDRs) from option b) or c), with the proviso that at least one of the CDRs has 66% or greater sequence identity to the respective SEQ ID NO:; wherein the CDRs are embedded in a suitable protein framework so as to be able to bind to human CD79b, or a target-binding fragment or derivative thereof that retains target-binding ability to human CD79b.

2. The antibody or fragment of claim 1 , wherein the antibody is a humanized antibody or fragment.

3. 3. The antibody or fragment of claim 1 or 2, a) SEQ ID NOs: 3 and 4; SEQ ID NOs: 13 and 14, SEQ ID NOs: 23 and 24, or a pair of heavy chain / light chain variable domains (HCVD / LCVD) as set forth in the pair of SEQ ID NOs: 33 and 34; b) a heavy chain / light chain variable domain (HCVD / LCVD) pair of a) with the proviso that: the HCVD has 80% or greater sequence identity to the HCVD contained in the respective SEQ ID NO: and / or LCVDs are pairs that have 80% or more sequence identity to the LCVDs contained in their respective SEQ ID NOs. c) A pair of heavy chain / light chain variable domains (HCVD / LCVD) according to option a) or b), wherein at least one of the HCVD or LCVD domains has up to 10 amino acid substitutions relative to the HCVD or LCVD domain contained in the respective SEQ ID NO:; The antibody or fragment thereof, which is capable of binding to human CD79b.

4. The antibody or fragment according to any one of claims 1 to 3, wherein at least one amino acid substitution is a conservative amino acid substitution.

5. the human CD79b to which the antibody or fragment binds is a) an amino acid sequence set forth in SEQ ID NOs: 41 to 44, or b) an amino acid sequence having 80% or more sequence identity with SEQ ID NOs: 41 to 44 The antibody or fragment according to any one of claims 1 to 4, comprising:

6. 6. The antibody or fragment of any one of claims 1 to 5, which is a monoclonal antibody, or a target-binding fragment or derivative thereof that retains target-binding ability to human CD79b.

7. IgG, scFv, Fab, or (Fab) ２ The antibody or fragment according to any one of claims 1 to 6, which is an antibody in at least one format selected from the group consisting of:

8. A nucleic acid encoding at least one chain of an antibody or fragment according to any one of claims 1 to 7.

9. A recombinant immunotoxin, immunocytokine, antibody drug conjugate or antibody-radionuclide conjugate comprising the antibody or fragment of any one of claims 1 to 8.

10. A pharmaceutical composition comprising the antibody or fragment according to any one of claims 1 to 7, the nucleic acid according to claim 8, or the recombinant immunotoxin, immunocytokine, antibody drug conjugate or antibody-radionuclide according to claim 9, and optionally one or more pharmaceutically acceptable excipients.

11. (i) an antibody or fragment according to any one of claims 1 to 7, a nucleic acid according to claim 8, a recombinant immunotoxin, an immunocytokine, an antibody drug conjugate or an antibody-radionuclide according to claim 9, or a pharmaceutical composition according to claim 10, and (ii) one or more therapeutically active compounds A combination comprising:

12. Use of an antibody or fragment according to any one of claims 1 to 7, a nucleic acid according to claim 8, a recombinant immunotoxin, immunocytokine, antibody drug conjugate or antibody-radionuclide according to claim 9, a pharmaceutical composition according to claim 10 or a combination according to claim 11 for the treatment of a human or animal subject diagnosed with, suffering from or at risk of suffering from a neoplastic disease, or for the prevention of such a condition (for the manufacture of a medicament).

13. A method for treating or preventing a neoplastic disease, comprising administering to a human or animal subject a therapeutically sufficient dose of the antibody or fragment according to any one of claims 1 to 7, the nucleic acid according to claim 8, the recombinant immunotoxin, immunocytokine, antibody-drug conjugate or antibody-radionuclide according to claim 9, the pharmaceutical composition according to claim 10 or the combination according to claim 11.

14. A medical kit of parts including: a) an antibody or fragment according to any one of claims 1 to 7, a nucleic acid according to claim 8, a recombinant immunotoxin, immunocytokine, antibody-drug conjugate or antibody-radionuclide according to claim 9, a pharmaceutical composition according to claim 10 or a combination according to claim 11, b) a device for administering the composition, composition or combination, and c) Instructions for use.

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