Humanized Anti-nuclear antibodies for targeting necrosis in cancer therapy

Abstract

To provide humanized monoclonal anti-nuclear antibodies with enhanced binding affinity and tumor uptake.SOLUTION: Particularly preferred antibodies are site-directed mutants of H-CDR3 with up to about 8-fold improvement in affinity as compared to the non-humanized non-mutated form. In further preferred aspects, such humanized antibodies are employed in tumor necrosis targeted delivery of immune modulators, immune effectors, and other therapeutic or diagnostic agents.SELECTED DRAWING: Figure 1C

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 473,554, filed March 20, 2017, the contents of which are incorporated herein by reference.

[0002] The field of the invention is antibodies, particularly humanized antibodies that target necrosis in tumors. [Background technology]

[0003] The background description includes information that may be useful in understanding the invention. None of the information described herein is an admission that it is prior art or relevant to the claimed invention, and any publications referenced specifically or by implication are not an admission that they are prior art.

[0004] All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. If a definition or use of a term in an incorporated reference contradicts or conflicts with a definition of that term set forth herein, the definition of that term set forth herein shall control and the definition of that term in the reference shall not be used.

[0005] Previously, monoclonal antibodies directed against universal intracellular antigens that become accessible after cell death have been used for the immunotherapy of solid tumors (see, for example, Cancer Research, 48:5842-5848, 1988; Hybridoma, 12(6):689-698, 1993; or Cancer Biotherapy & Radiopharmaceuticals, 13(4):255-68, 1998). Advantageously, such approaches target necrotic areas of tumors that are present in all tumors but are not usually present in normal tissues of the body. For example, three "tumor necrosis targeting (TNT)" mouse monoclonal antibodies have been studied, including muTNT-1, directed against histone H1 and DNA, muTNT-2, which binds to a common epitope on histone fractions H1 and H3, and muTNT-3, which binds primarily to single-stranded DNA. In particular, biodistribution analysis in tumor-bearing mice demonstrated that TNT-3 exhibited the highest absorption in tumors compared to muTNT-1 and muTNT-2, and TNT-3 has been proposed for radioimmunotherapy of solid tumors (see, e.g., Cancer Biother Radiopharm 1998 Aug;13(4):255-68). In an alternative approach, a chimeric TNT-3 antibody / mouse interferon-gamma fusion protein has been constructed for immunotherapy of solid malignancies (see, e.g., Hybrid Hybridomics 2003 Aug;22(4):197-207) and has demonstrated a variety of desirable properties.

[0006] Unfortunately, mouse monoclonal antibodies are immunogenic in humans, greatly limiting their therapeutic potential in human patients. To help address the issues associated with immunogenicity, chimeric TNT-3 antibodies were constructed using human constant regions that account for 65% of the molecule and the mouse variable regions. However, such chimeric antibodies still contained about 35% mouse residues, so the antibodies remained immunogenic.

[0007] To further reduce the immunogenicity of chimeric antibodies, various approaches can be undertaken. One of the simplest humanization approaches is complementarity determining region (CDR) grafting, in which six antibody CDRs are simply grafted into the corresponding human framework regions, which further reduces the number of mouse residues by about 25%. Unfortunately, while CDR grafting has been successfully used to humanize some antibodies, at least in some cases, grafting often alters the binding profile, resulting in an undesirable reduction in affinity to the antigen (see, for example, J Mol Biol 224:487-499, 1992), with other publications reporting reductions of up to several hundred fold (see, for example, Proc. Natl Acad. Sci. USA, 89(10):4285-9, 1992). Therefore, in most cases, CDR grafting must be combined with fine turning of some framework residues that are potentially important for restoring the affinity of the humanized antibody. However, the number of important framework residues that can potentially contribute to the CDR structure is relatively large and often hampers rational antibody design. Moreover, the interactions of framework residues with one or more CDRs further complicate the analysis, making the fine-tuning process generally time-consuming and unpredictable.

[0008] In yet another approach, the affinity of humanized antibodies was restored by stepwise mutation of the CDRs (see, e.g., Proc. Natl Acad. Sci. USA, 95, 6037-6042, 1998). Furthermore, some have reported that diversity in the CDR regions of the VH chain is sufficient for most antibody specificities, and have pointed out that somatic mutations can, surprisingly, allow high affinity. Indeed, H-CDR3 was shown to play a major role in antigen binding, as suggested by earlier studies. This shows that the heavy chain alone, or even a single VH domain, can bind antigen with affinity comparable to that of intact antibodies (see, e.g., Nature 341, 544-546, 1989; and J. Immunol. Methods 193, 177-187, 1996). Of course, it is possible that the selected CDR modifications may again change the overall configuration of the binding site, once again introducing a high level of unpredictability. Summary of the Invention [Problem to be solved by the invention]

[0009] Thus, there is a need for humanized antibody compositions and methods that allow targeting of the tumor microenvironment, particularly necrotic cells, to allow site-specific modulation of the immune response against tumors. [Means for solving the problem]

[0010] The present subject matter relates to compositions of humanized TNT antibodies with improved binding characteristics, methods for their production, and uses.

[0011] In one aspect of the inventive subject matter, the inventors contemplate humanized antinuclear antibodies that have improved binding affinity to nuclear targets (e.g., DNA, especially single-stranded DNA (ssDNA)) compared to the corresponding non-humanized antibody. In particular, the contemplated humanized antibodies have binding affinity at least 2-fold, more typically at least 4-fold, and most typically at least 8-fold higher than the corresponding non-humanized antibody. Furthermore, preferred humanized antibodies have mutated amino acids in the H-CDR3 relative to the corresponding non-humanized antibody. For example, the mutated amino acids in the H-CDR3 may be 107Phe→Arg, 101Ile→His, 102Gly→Arg, 109Tyr→Thr, 103Val→Ser, 104Arg→Thr, 109Tyr→Arg, and 104Arg→Leu.

[0012] Viewed from a different perspective, the humanized antibody may have at least one of an H-CDR1 sequence comprising TRYWMH (SEQ ID NO: 1), an H-CDR2 sequence comprising GAIYPGNSDTSYNQKFKG (SEQ ID NO: 2), an H-CDR3 sequence comprising EEIGVRRWFAY (SEQ ID NO: 3), an L-CDR1 sequence comprising RARQSISNYLH (SEQ ID NO: 4), an L-CDR2 sequence comprising ASQSIS (SEQ ID NO: 5), and an L-CDR3 sequence comprising QQSNSWPLT (SEQ ID NO: 6). Thus, in a further contemplated aspect, the humanized antibody may have a VH sequence comprising: [ka] and / or a VL sequence comprising: [ka] may have

[0013] In another aspect of the subject matter of the present invention, the inventors also contemplate hybrid molecules comprising at least a binding portion of the humanized antibody contemplated herein, which binds a therapeutic agent and / or an imaging agent. For example, suitable binding portions of antibodies include full-length antibodies, Fab fragments, F(ab')2 fragments, Fab2 fragments, and scFvs, while contemplated therapeutic agents include cytokines or portions thereof, chemokines or portions thereof, inhibitors of myeloid-derived suppressor cells (MDSCs), inhibitors of M2 macrophages, radioisotopes, costimulatory molecules, Toll-like receptor ("TLR") agonists or ligands, molecules that interfere with epithelial-mesenchymal transition ("EMT"), and / or chemotherapeutic drugs. Contemplated imaging agents include radioisotopes, positron emission tomography (PET) labels, and / or single photon emission computed tomography (SPECT) labels. Thus, the inventors also contemplate pharmaceutical compositions comprising the antibodies or hybrid molecules presented herein. Most typically, such pharmaceutical compositions will be formulated for injection.

[0014] In yet another aspect of the inventive subject matter, the inventors contemplate a method of targeting necrotic cells, which would comprise the step of contacting necrotic cells with an antibody or hybrid molecule as set forth herein. Typically, but not limiting to the inventive subject matter, it is preferred that the necrotic cells are necrotic tumor cells within a tumor microenvironment and / or that the contacting step is performed in vivo.

[0015] Thus, the inventors also contemplate methods of delivering a therapeutic or imaging agent to a tumor microenvironment containing necrotic tumor cells. In such methods, a therapeutic or imaging agent is provided that is conjugated to an antibody as set forth herein. The necrotic tumor cells are then contacted within the tumor microenvironment with the therapeutic or imaging agent under conditions that allow the antibody to bind to nuclear targets within the necrotic cells within the microenvironment.

[0016] In a preferred aspect, the therapeutic agent comprises at least one of a cytokine or a portion thereof, a chemokine or a portion thereof, an inhibitor of MDSC, an inhibitor of M2 macrophage, a radioisotope, a costimulatory molecule, a TLR agonist or ligand, a molecule that prevents EMT, and a chemotherapeutic agent, while the imaging agent comprises at least one of a radioisotope, a PET label, and a SPECT label. Most typically, the tumor is a solid tumor. And the delivery of the therapeutic agent or imaging agent may be enhanced using a vascular permeability enhancer (e.g., IL-2).

[0017] In a further aspect of the subject matter of the present invention, the inventors further contemplate a method for generating affinity-optimized humanized antibodies derived from murine antibodies. Most typically, such a method will comprise the step of identifying a human antibody sequence having a predefined minimum identity with the murine antibody sequence (e.g., at least 90%, 92%, 94%, or 96%, excluding the CDRs). In another step, the murine CDRs for VH and VL are grafted into the VH and VL of the identified human antibody sequence, respectively, to obtain chimeric VH and VL sequences, and scFv-hybrids are generated with the chimeric VH and VL sequences. Site-directed mutagenesis is then performed on the H-CDR3 to generate an scFv-hybrid library, and affinity selection (and / or maturation) is used to identify binders from the scFv-hybrid library. In yet another step, the chimeric VH and VL sequences of the binders are grafted into human antibody sequences to obtain affinity-optimized humanized antibodies.

[0018] Most typically, the scFv-hybrid is a phage display hybrid, but may also be an RNA display hybrid. Furthermore, it is generally preferred (but not essential) that the site-directed mutagenesis in the H-CDR3 is directed to a single amino acid position in the H-CDR3. Similarly, it is generally preferred (but not essential) that the affinity selection is carried out over at least three successive selection / enrichment cycles. If desired, the affinity selection may further include at least one step of additional site-directed mutagenesis in the H-CDR3 and / or framework regions.

[0019] Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments, taken in conjunction with the accompanying drawings in which like reference numerals represent like elements. [Brief description of the drawings]

[0020] [Figure 1A-1C] Schematic diagrams showing exemplary alignments of VH (FIG. 1A) and VL (FIG. 1B) sequences of human, mouse, and humanized protein moieties, as well as the general assembly of exemplary EP / scFv (FIG. 1C). In FIG. 1A, mTNT-3 / HV is SEQ ID NO: 9, EP / HV is SEQ ID NO: 10, and HuFdl38 / HV is SEQ ID NO: 11. In FIG. 1B, mTNT / LV is SEQ ID NO: 12, EP / LV is SEQ ID NO: 13, and huB-B10 / LV is SEQ ID NO: 14. [Diagram 2] Mutations of H-CDR3 of EP / ScFv and its panning of the library against crude DNA are shown. The EEIGVRRWFAY (SEQ ID NO: 3) domain is the heavy chain CDR3 sequence. The shaded "X" can be any one of the 20 naturally occurring amino acids. Primers Pr1-Pr11 are SEQ ID NOs: 16-26, respectively. [Diagram 3] 1 is a graph showing an exemplary increase in binding over successive rounds of panning. [Figure 4]1 is a plot showing the results of binding phagemid clones. [Diagram 5] Photograph of an SDS gel of an exemplary humanized IgG sample. [Figure 6] 1 is a graph showing exemplary relative affinities of selected mutant antibody proteins, EP27, EP7, EP35, EP103, EP54, EP102, and EP51. [Figure 7] 1 shows various photomicrographs of immunostaining of selected humanized antibodies in accordance with the subject matter of the present invention. [Figure 8] Graph showing affinity measurements for EP2 and EP51 humanized antibodies. [Figure 9A-9D] 1 shows exemplary graphs of the biodistribution of various humanized antibodies across different tissues and time points in accordance with the present inventive subject matter. [Figure 10] 1 shows an exemplary graph of the biodistribution of one humanized antibody in accordance with the present inventive subject matter across various tissues without enhanced vascular penetration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The inventors have now discovered that antibodies against a variety of targets can not only be humanized, but also conceptually and effectively improved with respect to antigen affinity. For example, and as described in more detail below, the inventors have converted a murine antinuclear antibody into a humanized antibody with substantially improved affinity.

[0022] More specifically, and based on the desired mouse antibody (e.g., a mouse antibody that binds ssDNA), the corresponding human antibody sequence with the highest amino acid identity was identified in an antibody database. Most typically, suitable human antibody sequences will have a given minimum identity of at least 90%, at least 92%, at least 94%, or at least 96%, excluding the CDRs. Once identified, the mouse CDRs for VH and VL are then grafted into the human sequence to obtain chimeric VH and VL sequences using conventional methods well known in the art. Of course, it should be understood that such grafting steps can be done in vitro or in silico if an actual chimeric sequence is not required for the experiment. Regardless of the method of grafting, scFv-hybrids are then generated using the chimeric VH and VL sequences as a base structure for affinity maturation or other improvements, if desired. Again, there are numerous ways to produce scFvs from actual nucleic acid moieties or sequence information known in the art, and all methods of generating such scFvs are considered suitable for use herein. In a particularly preferred aspect, the scFv is fused to a selection system that allows for rapid identification and clonal amplification of variants with the desired affinity or other molecular properties. For example, selection systems suitable for use herein include various phage panning systems (e.g., based on pIII, pVIII, pIX) and RNA display systems (e.g., puromycin binding-based), and it should be understood that all known methods of affinity maturation and isolation of selected closes are considered suitable for use herein. Thus, clonal selection and / or affinity maturation may include one or more steps of intermittent site-directed mutagenesis targeting at least one of the CDRs and / or framework regions.

[0023] In a further aspect of the subject matter of the present invention, the inventors have noted that affinity and even bioavailability / absorption can be improved by modifying only the H-CDR3 portion. In particular, the inventors have performed a single amino acid scanning run and found that selected mutants have substantially increased binding, as also shown in more detail below. Advantageously, such mutagenesis can be done using the scFv format, rather than testing full-length antibodies (typically IgG). As will be readily understood, the affinity-improved chimeric VH and VL sequences of the scFv can then be grafted into human antibody sequences, resulting in affinity-optimized humanized antibodies. The prepared humanized affinity-improved antinuclear antibodies were then tested in various assays to confirm proper and selective binding, as also shown in more detail below.

[0024] With regard to the intended use of the antibodies thus prepared, it should be understood that the antinuclear antibodies presented herein are particularly useful for targeting necrotic, and in particular, necrotic cells within the tumor microenvironment. Because the tumor microenvironment, which promotes various mechanisms of immune evasion, is often a difficult-to-target environment (e.g., hypoxia, which reduces the activity of NK cells, nutrient and oxygen deprivation, which promotes EMT, etc.), specific delivery and retention of various immune stimulators to the microenvironment may be particularly beneficial for immunotherapy.

[0025] In that regard, it should be understood that the terms "apoptosis" and "necrosis" are not used interchangeably herein and refer to two main distinct mechanisms and pathways of cell death. Apoptosis is a well-defined process of programmed cell death that includes specialized signaling events and stepwise cell closure (e.g., blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, DNA fragmentation, mRNA decay), while necrosis is typically manifested as a disruptive process of cell death with concomitant loss of organelle function, cell destruction, and release of cellular contents into the environment. Furthermore, necrosis is typically accompanied by an inflammatory cellular response. Furthermore, it should be understood that necrosis is the site where the immune system "sees" and immunologically reacts to the tumor. This is important because delivery of payloads to necrosis will help the immune system recognize and respond to the tumor, making it a preferred site for delivery of therapeutically effective payloads.

[0026] Thus, the inventors also contemplate the use of antibodies and fragments thereof to deliver imaging and / or therapeutic agents to the tumor microenvironment. Most typically, the antibodies or fragments thereof will be specific in binding to nuclear targets, and in particular to ssDNA (i.e., have a Kd of 10 or greater, as determined, for example, by surface plasmon resonance (SPR) or other techniques). -7 Less than M, more typically 10 -8 Most typically, but not necessarily in all cases, binding to ssDNA will be independent of the presence of histones and other nuclear proteins.

[0027] Of course, it should be understood that the imaging agent and / or therapeutic agent may be attached to the antibody (or fragment thereof) in many ways, including covalent and non-covalent. Most typically, the attachment will be covalent, which may be achieved using conventional coupling chemistries, such as amino group reactive reagents (e.g., N-hydroxy-succinimide esters, various aldehydes, carbodiimide compounds, epoxides, imidoesters, etc.), or sulfhydryl group reactive reagents (e.g., various maleimides, thiols, etc.), or may be carried out via recombinant cloning techniques, in which the antibody (fragment) is fused in frame to an optional linker that is fused in frame to a second protein of interest. An appropriate linker may be selected according to the desired length (e.g., a length that provides a desired spatial distance), amino acid composition (e.g., a length that provides a cleavable or flexible linker), etc. In further contemplated embodiments of the binding, the binding may be non-covalent, and in particularly preferred ways, the binding is provided by elements of known binding pairs, such as biotin / avidin, cellulose / cellulose binding protein, nickel-nitrilotriacetic acid (Ni-NTA) / oligo-histidyl, etc.

[0028] With respect to diagnostic agents, it is intended that all detectable (and preferably quantitatively detectable) agents are considered suitable for use herein. It should be noted further that detection can be performed ex vivo (e.g., on tissue sections) and / or in vivo using suitable methods known in the art. For example, visually detectable imaging agents include fluorophores, luminescent groups, catalytically active groups (e.g., catalytically active groups that precipitate dyes and / or activate chromogens or luminogens), radiographically detectable groups (e.g., PET, SPECT, NMR labels, radioisotopes, etc.).

[0029] Similarly, and with regard to therapeutic agents, it is intended that all therapeutic agents are considered suitable for use herein. However, in a particularly preferred embodiment of the subject matter of the present invention, the therapeutic agent will have an immune stimulatory effect. Most typically, the effect of such stimulators will be to cancel or neutralize one or more mechanisms leading to immune evasion of cancer cells within the tumor microenvironment. For example, when immune evasion is based on the recruitment of M2 macrophages or regulatory T cells (Tregs), suitable therapeutic agents will include those that specifically inactivate or destroy such inhibitory cells (e.g., gemcitabine, RP-182 (see SEQ ID NO: 121 in US Pat. No. 9,492,499), or cyclophosphamide). Additionally or alternatively, when immune evasion is based on checkpoint inhibition by effector and / or helper cells, binders or antagonists against CTLA4 or PD1 (e.g., ipilimumab, pembrolizumab, etc.) are contemplated.

[0030] Conversely, it should also be understood that the use of a therapeutic agent may enhance immunotherapy if the therapeutic agent has immune stimulatory activity. Such immune stimulatory activity may be achieved through the use of costimulatory signals coupled to the antibody or fragment thereof, preferably in the context of one or more tumor (neo)antigens. For example, costimulatory signals include 4-1BBL, OX40L, GITRL, TIM3, LFA3, ICAM1, ICOSL, and others. It should also be understood that immune stimulatory agents would include immune stimulatory cytokines, such as IL-2, IL-12, IL15, IL-15 superagonists, TLR agonists, and ligands. It should also be understood that a therapeutic agent may include (proinflammatory) chemokines, which would further attract immune competent cells. In this context, it should be understood that the therapeutic agent will typically not kill tumor cells at the site of necrosis (because necrotic cells are no longer viable), but will promote an enhancement of the immune response against tumor cells proximal to the necrotic tissue (e.g., by utilizing antigen spreading and antigen cascading, or by the reversal of immune suppressive mechanisms).

[0031] If desired, the therapeutic agent may include an agent that will target factors that contribute to EMT (epithelial-mesenchymal transition) within the tumor microenvironment, including IL-8 and TGF-β. Thus, suitable therapeutic agents will also include those that bind to or otherwise sequester IL-8 and TGF-β.

[0032] In addition, therapeutic agents may include more conventional drugs used in the treatment of cancer. For example, typical anti-cancer drugs include antimetabolites, drugs that interfere with the formation or disassembly of microtubules, DNA alkylating agents, and topoisomerase inhibitors, cytotoxic drugs, and others, all of which may be cleavable under conditions prevalent in the tumor microenvironment. Also contemplated therapeutic agents include radiotherapeutic agents, such as alpha-emitters and beta-emitters (e.g., Bi-213, Pb-212, I-131, Ac-225, Sr-89, and others). Such agents will generally affect non-necrotic tumor cells within the tumor or tumor microenvironment.

[0033] Consequently, and as set forth in more detail below, the inventors contemplate methods of targeting necrotic cells, which generally include contacting necrotic cells (typically tumor cells, most typically necrotic tumor cells within a tumor microenvironment) with a binding agent that specifically binds to nuclear targets, and in particular to ssDNA. Moreover, the contacting may be performed in such methods in vivo or in vitro. For example, when the process is performed in vitro, a relatively small amount (e.g., 0.001-100 μg, 0.01-0.1 μg, or 0.001-0.01 μg) of antibody may be required. On the other hand, when the process is performed in vivo, a relatively large amount (e.g., 0.01-100 mg, 0.1-10 mg, or 1-10 mg) of antibody may be required. Of course, it should be understood that when the antibody is conjugated to an imaging agent and / or a therapeutic agent, the amount of antibody will be determined, at least in part, by the type and amount of imaging agent and / or therapeutic agent required for the desired effect.

[0034] Consequently, the inventors also contemplate methods of delivering therapeutic and / or imaging agents to a tumor microenvironment containing necrotic tumor cells. As noted above, such methods will typically include the steps of providing a therapeutic or imaging agent that is bound to an antibody that binds to a nuclear target (and in particular ssDNA), and the further step of contacting (preferably in vivo) necrotic tumor cells in the microenvironment with the therapeutic or imaging agent under conditions in which the antibody can bind to the nuclear target in the necrotic cells within the tumor microenvironment.

[0035] Additionally, the methods contemplated herein may further comprise one or more steps to enhance tumor necrosis, thereby enhancing absorption of the modified antibody into the tumor, thereby optimizing delivery of the therapeutic or diagnostic payload. For example, suitable further steps include radiation therapy, chemotherapy, or immunotherapy, and especially low-dose metronomic chemotherapy and radiation therapy. EXAMPLES

[0036] Reagents: Plasmids pEE6hCMV-B and pEE12 carrying the glutamine synthetase gene amplification system were purchased from Lonza Biologies (Slough, UK). Restriction enzymes, T4 DNA ligase, and other molecular biology reagents were purchased from New England Biolabs (Beverly, MA) or Boehringer Mannheim (Indianapolis, IN). RPMI-1640 medium, MEM non-essential amino acid solution, penicillin-streptomycin solution, digested crude DNA, ABTS (2,2'-azobenzene-2-carboxylic acid) were purchased from Sigma Chemical Co. (St. Louis, MO). Hybridoma serum-free medium (SFM medium) (with and without glutamine) was purchased from Life Technologies, Inc. (Gaithersburg, MD), and dialyzed fetal bovine serum was obtained from HyClone Laboratories, Inc. (Logan, UT). Iodine-125 was obtained from DuPont / New England Nuclear (North Billerica, Mass.) as sodium iodide in 0.1 N sodium hydroxide. Six-week-old BALB / c mice were purchased from Harlan-Sprague Dawley (Indianapolis, Ind.).

[0037] Design and Assembling of EP / ScFv: By searching the human antibody database, two sequences, huFd138 HV and hu B-B10 LV, were found to be most identical to the original TNT-3 amino acid sequences of the heavy and light chains, respectively. Therefore, they were used as master templates to construct the EP variable heavy and variable light chains, respectively. The six CDRs were then simply grafted into the corresponding human framework regions. The six mouse TNT-3 CDR sequences were converted to the corresponding nucleotide sequences using the codon usage observed in known human antibodies. The sequences of the variable heavy and variable light chains were combined with a (GSSSS)3 linker to construct the EP / ScFv. The designed DNA sequence was split into eight overlapping oligonucleotides and synthesized.

[0038] The full-length ScFv was obtained by serial assembly PCR and amplified with a pair of primers at the 5' and 3' ends. The PCR product was digested with NotI and SpeI and cloned into pBlueScript SK(-). One clone with the correct sequence was selected by DNA sequencing as a standard template for the next mutation. To restore and improve the affinity of the EP antibody candidate (also referred to herein as EP, huTNT IgG1, or antibody), a site-specific random mutation on the H-CDR3 domain encompassing 11 codons was performed. A set of primers containing 11 site-specific mutations was designed. Using PCR, random mutations of 11 amino acid residues were introduced stepwise. The 11 mutated humanized ScFvs were amplified by PCR with a pair of primers at the 5' and 3' ends. The PCR product was then purified, digested with NotI and SpeI, and inserted into the digested lambda phage SurfZAP TM vector. The packaged SurfZAP™ vector infected into host cells XL-1 was then amplified and excised into phagemid pSurfscript™ SK(-) by ExAssist™ help phage. The phagemids propagated in SOLR cells to eliminate the help phage were amplified in XL-1 cells. EP / ScFv was expressed as a fusion protein (ScFv / cpIII) and incorporated into the coat protein of recombinant phagemid particles by VCSM13 helper phage. Phagemids were collected from the amplified supernatant by PEG precipitation and concentrated.

[0039] Four rounds of biopanning on crude DNA coated onto 25 ml flasks were performed according to the STRATAGENE protocol. Briefly, for each panning, >10 12phagemids were used. Unbound phage were removed with 10 PBST washes and bound phage were eluted with 5 ml glycine-HCl (pH 2.2). The titer of eluted phage was determined by plating 100 μl of infected bacteria on selection plates and counting colony forming units (cfu). ScFv phage from the fourth round of panning were tested for reactivity with crude DNA by phage-ELISA with 100 μl of 10 μg / ml crude DNA coated on each well of an ELISA plate. Bound phage were detected with rabbit anti-M13 antibody followed by goat anti-rabbit antibody horseradish peroxidase conjugate. A 405 Values ​​were determined after 10 min incubation with substrate (ABTS) and measured by an ELISA reader.

[0040] Construction and expression of EP IgG: To recover the intact antibody molecule, the EP / ScFv was converted to EP IgG1. To achieve this, the EP heavy and light chain variable regions were PCR amplified from the cloning vectors using primers designed to introduce appropriate restriction enzyme sites. The heavy chain variable region was introduced into the expression vector pEE6hCMV-B into which the human gamma 1 constant region (CH) had been previously cloned. The light chain variable region was inserted into the expression vector pEE12, which contains the human VL constant region cDNA. The final expression vectors were pEE6hCMV-B / EP HC and pEE12 / EP LC, which contain transcription cassettes for the human light and heavy chains, respectively, under the control of the CMV major immediate early promoter. pEE12 / EP LC also contains the selectable marker, the cDNA sequence for glutamine synthetase, under the control of the SV40 early promoter. After completion of each vector, pEE6hCMV-B / EP HC and pEE12 / EP LC vectors were co-transfected into host NS0 mouse myeloma cells for expression of EP IgG1. Supernatants containing antibody product were initially identified by indirect ELISA using 96-well microtiter plates coated with degraded crude DNA at 10 μg / ml. The highest producing clones were identified by a 24-hour productivity assay. After two rounds of subcloning by limiting dilution, the highest producing clones were expanded and EP IgG1 was stepwise purified from cell culture supernatants by protein A affinity and ion exchange chromatography. The purity of the EP antibody was examined by SDS-PAGE.

[0041] Determination of crude DNA binding by ELISA: To measure the DNA binding activity of EP IgG candidates, 96-well ELISA plates were coated with 100 μl of 10 μg / ml crude DNA, blocked with 1% BSA-PBS, and serial dilutions of EP IgG candidates were added to the wells and incubated at 37° C. for 1 hour. Purified chTNT-3 was used as a standard. Any unbound antibody was removed by triplicate washes with 0.05% PBST before the addition of a goat anti-human IgG secondary antibody conjugated to horseradish peroxidase followed by a colorimetric substrate. OD 405 was measured by an ELISA reader.

[0042] Localization of EP candidates in Raji cells by indirect immunofluorescence staining: In order to observe the localization of EP IgG candidates in Raji cells, indirect immunofluorescence staining was performed. Briefly, Raji cells were fixed with 10% buffered neutral formalin and washed with PBS. 104 Raji cells in 200μl PBS were reacted with various EP antibodies for 1 hour at 37℃ in a 1.5ml tube. The cells were washed five times with PBST and unbound antibodies were removed by centrifugation. FITC-conjugated goat anti-human IgG antibody was added at a dilution of 1:30, and finally the Raji cells were collected in 20μl PBS, spread on a slide and observed under a fluorescent microscope.

[0043] Preparation of iodine-125 labeled MAbs: Iodine-125 labeled MAbs were prepared using a modified chloramine-T method. Briefly, 1 mCi of I 125 and 20 μl of an aqueous solution of chloramine-T (2 mg / ml) was added to a 5 ml tube containing 100 μg MAb in 100 μl PBS. The solution was quenched after 2 min with 10 μl of an aqueous solution of sodium metabisulfite. Each reaction mixture was then purified using a Sephadex G-25 column, typically resulting in a recovery of 90%-95%. Radiolabeled MAbs were diluted in PBS for injection, stored at 4° C., and administered within 2 hours after labeling.

[0044] Binding and biodistribution studies: In vitro immunoreactivity of radiolabeled MAbs was assessed by conventional fixed cell radioimmunoassay using Raji cells. Briefly, radioiodinated MAbs (approximately 100,000 cpm / tube) were injected into 10 6 The EP antibodies were incubated with 100 fixed Raji cells in triplicate for 1 h with intermittent mixing. After incubation, the cells were washed three times with 1% BSA in PBS. Bound MAb was detected by measuring the radioactivity associated with the cell pellet in a gamma counter. Fixed Raji Burkitt lymphoma cells were used as antigen cells to determine the binding avidity constant (Ka) of the EP antibodies. Various concentrations (10-110 ng) of the EP antibodies were 125 I-labeled EP antibody was 6 After incubation with fixed Raji cells at room temperature with constant mixing, and removing unbound radioactivity by washing three times with PBST, the amount of bound radioactivity was counted in a gamma counter and used to calculate the binding avidity constant (Ka) by Scatchard analysis. The binding avidity constant Ka was determined by K=-(slope / 2).

[0045] To investigate the biodistribution of EP antibody and chTNT-3, 10 7 A 0.2 ml inoculum containing LS 174T human colon adenocarcinoma cells was injected sc in the left flank. Tumors were allowed to grow until they reached approximately 1 cm in diameter. Within each group (n=5), individual mice received 100 μCi / 10 μg of 125 0.1 ml inoculum containing I-labeled MAb was injected iv. At various post-injection time points, mice were sacrificed and organs, blood, and tumors were removed and weighed. Radioactivity in samples was then measured and expressed as %ID / g and tumor / organ ratio (cpm per gram tumor / cpm per gram organ). Significance levels were determined using the Wilcox rank sum test.

[0046] Design and Construction of Humanized TNT-3 / ScFv: To design the humanized TNT-3 framework of the V region, we searched the human antibody database and found that two human antibodies, huFd138 HV and hu B-B10 LV, were most identical to the original TNT-3 amino acid sequences of the heavy and light chains, respectively. huFdl38 HV had 81% homology of frameworks 1, 2, 3, and 4 to the mTNT-3 heavy chain sequence (see FIG. 1A); hu B-B10 LV had 74% homology of frameworks 1, 2, 3, and 4 to the mTNT-3 light chain sequence (see FIG. 1B). Three residues (I, II, III, IV, IV, IV, V ... 48 , T 74 , and Y 95 ) were retained. The original sequences of the six complementarity determining regions (CDRs) of the heavy and light chains were maintained, and all other sequences were replaced by human sequences. The EP candidate variable heavy and light chain sequences were combined with a (GSSSS)3 linker to construct the EP / ScFv (see FIG. 1C). Further with reference to FIGS. 1A-1B, amino acid sequence alignments of mouse TNT-3 and human TNT-3 templates are shown. FIG. 1A shows the alignment of mouse TNT-3 (mTNT-3), huFd138 HV, and the EP heavy chain; FIG. 1B shows the alignment of mTNT-3, hu B-B10 LV, and the EP light chain templates. The numbering of each human template is described in the text. The framework and CDR sequences of the light and heavy chains according to the Kabat definition are labeled. * indicates residues replaced with human amino acids; # indicates mouse residues that were maintained. Figure 1C shows the schematic HV and LV constructed with (GSSSS)3 linker in EP / ScFv. Notably, only EP / ScFv with 6 CDRs simply grafted into human framework showed very weak reaction to crude DNA by phage-ELISA. This indicated that such substitution of framework caused a large decrease in EP / ScFv affinity.

[0047] Mutation of H-CDR3 of EP / ScFv and panning of the library against crude DNA: To improve the affinity of EP / ScFv, site-directed random mutations on the H-CDR3 domain were performed with 11 primers, each containing the randomly mutated codons shown in Figure 2, which were sequentially introduced into the heavy chain by PCR to generate 11 sub-libraries of mutated ScFv.

[0048] The 11 sublibraries were mixed together equally and inserted into the digested lambda phage SurfZAP™ vector. The EP ScFv was digested with SpeI and NotI and inserted into the lambda phage SurfZAP vector, then excised and placed into the pSurfscript SK(-) vector. This resulted in the EP / ScFv being expressed as a fusion protein (ScFv / cpIII) to generate a general library with a diversity of 352. For panning, this library was split into 10 12 The phage-displayed EPs were selected and enriched by elution from DNA-coated flasks. After each panning run, the supernatants were incubated in 96-well ELISA plates precoated with DNA. After washing, HRP-conjugated anti-M13 antibodies were added and plates were read at 405 nm using ABTS as substrate). Library binding after 3 rounds of panning was 4-fold higher than the original. More than 90% of the recovered clones after 4 rounds of panning were positive for DNA binding (Figure 4). Phagemid particles were prepared from clones by PEG precipitation and resuspended in 1% Blotto / TB blocking buffer. 10 in 100 μl 9Each sample of cfu was added to an ELISA plate well coated with crude DNA. Unbound phagemids were removed by washing with PBST. Bound phagemids were detected with anti-M13 polyclonal antibody followed by HRP goat anti-rabbit IgG. Enzyme activity was measured at 405 nm using ABTS as substrate), indicating a high enrichment process.

[0049] Construction, expression, and purification of EP IgG: Based on the phage ELISA screening results from four rounds of panning, OD 405 Sixteen clones with the highest values ​​were selected and sequenced. Sequencing analysis showed that all 16 clones contained eight different nucleotide and amino acid sequences within the H-CDR3. The determination of clone binding based on phage ELISA was limited by the purification and the amount of phage containing the ScFv fusion protein. Monovalent ScFvs, which have 8-10 times lower affinity than bivalent IgGs, would amplify this disadvantage. To further evaluate the differences between affinities, to understand the relationship between binding and site mutations on the H-CDR3, and to restore the capacity of the whole IgG antibody, all eight mutant ScFvs were converted to the corresponding IgG1 expressed in a glutamine synthetase gene amplification system. The clones with the highest yield were selected by a 24-hour ELISA assay. When the cell lines were grown in 1L bioreactors, the supernatant containing EP IgG1 was collected and purified stepwise by protein A affinity chromatography. Approximately 10 μg / ml of Ep IgG was obtained after purification. SDS-PAGE assay demonstrated that the eight mutants of EP IgG1 (referred to herein as EP or huTNT IgG1) were properly assembled (Figure 5: lanes (a) Low Range Marker (BioRad); (b) EP2; (c) EP7; (d) EP27.1; e.EP35.2; (f) EP51; g EP54; (h) EP102; (i) EP103; (j) chTNT-3). Two bands were observed at approximately 25 and 55 kD, which corresponded to the molecular weights of the light and heavy chains of Ep IgG1, respectively.

[0050] Assay of affinity of EP mutants and characterization of mutations: To measure whether the binding activity of EP IgG1 candidates was improved, we used ELISA to detect the binding activity to crude DNA. As can be easily seen from Figure 6, all eight candidates had a significantly improved affinity (1.45 to 8 times) compared to chTNT-3. Here, to measure the relative affinity of EP mutants binding to crude DNA, various concentrations of purified EP IgG1 were tested by ELISA. Dose-dependent response curves were obtained and the concentration at 0.3 OD was determined by the best-fit linear equation (purified chTNT-3 was used as a reference). The relative affinity was expressed by the ratio of EP concentration / chTNT-3 concentration at 0.3 OD.

[0051] Combinatorial affinity and mutation analysis showed that the two best affinity candidates, EP / m7 and EP / 27.1, obtained approximately 8-fold improvements (corresponding mutations were m7 Ile→His and 27.1 Gly→Arg) (see Table 1). Two other clones, EP / 35.2 and EP / m103 (where 35.2 Tyr and m103 Arg were changed to Thr and Leu, respectively), obtained a 4-fold increase in affinity. The remaining four clones, m54, m102, m2, and m51 (where changes were m54 Arg→Leu, m102 Tyr→Arg, m2 Phe→Arg, and m51 Val→Ser, respectively), also showed affinity increases ranging from 1.46 to 2.34-fold.

[0052] [Table 1]

[0053] Analysis of the amino acid profile on the H-CDR3 of the affinity-improved mutants showed that positively charged amino acids such as Arg and His replaced existing residues in four of the mutants, while the other three mutants showed substitutions with amino acids containing hydroxyl groups. These results implied that changes in the electrostatic or hydrogen-bonding capacity of the residues contributed to the improvement observed in antibody binding to crude DNA. In one exceptional case (m103), substitution of Arg with Leu also increased binding activity by 4-fold.

[0054] Immunofluorescence microscopy studies: In order to observe the specificity of the EP IgG candidates for antigen Raji cells, indirect immunofluorescence staining was performed. As shown in Figure 7, all EP IgG candidates stained fixed Raji cells, giving a nuclear pattern of staining. EP2, 7, 27.1, 35.2, 51, 102, and 103 produced prominent nuclear rim staining similar to chTNT-3; only EP54 did not produce a homogenous nuclear staining pattern. Neither cytoplasmic nor cell surface staining was observed in all preparations. Using equivalent doses, the order of strongest immunofluorescence staining intensity was 51, 2, chTNT3>7, 27.1, 54, 102>103, and 35.2. This is quite different from the results of binding to crude DNA. EP7 and 27.1, which had the highest affinity for crude DNA in the ELISA studies, showed no corresponding reaction with fixed Raji cells, whereas EP51 and 2, which had relatively low affinity for crude DNA, interestingly yielded the most intense staining patterns.

[0055] Determination of EP binding avidity constants (Ka) by RIA: Based on the results of immunofluorescence staining, the binding avidity constants (Ka) of the two Ep IgG candidates (EP51 and EP2) that showed the highest affinity to Raji cells were determined by RIA. Both EP IgG candidates were 125 The binding force constants were calculated using the bound radioactivity after labeling with I and incubating with fixed Raji cells (Figure 8). The binding force constants for EP51 and EP2 were 2.8 x 10 9 M -1 and 2.1 x 10 9 M -1The binding force constants of EP51 and EP2 were 1.4 × 10 9 M -1 The K values ​​of EP51 and EP2 were consistent with their responses to crude DNA and antigen Raji cells and were similar to those of chTNT-3.

[0056] Figures 9A and 9C show biodistribution studies in colon 26-mice using I-125 labeled humanized antibody, biodistribution after 3 days, and Figures 9B and 9D show biodistribution after 7 days. Data are shown as injected dose / gram and normal organ to tumor ratio (n=5 mice) after various time points.

[0057] Biodistribution analysis of EP51 humanized antibody in LS174T colon cancer nude mice (n=5) showing specific and stable uptake in tumors at 3, 5, and 7 days is shown in Figure 10. In comparison, normal organs showed reduced uptake of the labeled antibody, indicating clearance from the blood pool and no off-target binding. Biodistribution analysis further showed that EP51 has similar biodistribution properties to chTNT-3, as shown in Table 2.

[0058] [Table 2]

[0059] The percent tumor uptake of EP51 in the LS174T model was 5.90 and 4.97 by days 2 and 5, respectively, comparable to chTNT3 (5.62 and 4.28). Percent blood uptake was also very similar in amount. However, percent tumor uptake of EP2 appeared to be significantly lower than chTNT-3, despite its Ka being 1.5-fold higher than chTNT-3. By days 2 and 5, percent tumor uptake of EP2 in the LS174T tumor model was 2.69 and 1.33, respectively, which are significantly lower than chTNT-3. Further biodistribution studies shown in FIG. 9 for the EP27.1 and EP35.2 subclones, and in FIG. 10 for EP51, demonstrated enhanced uptake in the tumor, particularly for EP51, compared to both chTNT-3 and the fully human TNT antibody NHS76. Impressively, EP51 showed only a slight decline in the % injected dose / gram from days 3 to 7, demonstrating that the human antibody was significantly retained by the tumor, while uptake in normal tissues lost reactivity due to negative binding and clearance from the blood.

[0060] As can be seen from the previous data, only the initial humanized TNT-3 construct by framework replacement followed by grafting of six CDRs showed slightly detectable affinity, even though the sequences of the humanized frameworks in HV and LV were 81% and 74% identical to mTNT3, respectively. This finding suggests that humanization by simple grafting of six CDRs altered the original conformation of the CDRs. To restore affinity, site-directed random mutations of H-CDR3 were introduced by a set of primers. A library of EP ScFvs with mutated H-CDR3 was displayed on lambda phage. After four rounds of selection on crude DNA, eight mutants with significantly higher affinity were efficiently enriched compared to the marginal detectable affinity of the initial humanized TNT-3.

[0061] ELISA studies of binding crude DNA showed that all eight mutants showed much higher affinity (1.45 to 8.25-fold) than the parent antibody chTNT-3. Analysis of H-CDR-3 sequences showed that most of the mutations with high affinity were substituted with positively charged amino acids, such as Arg and His, or amino acids containing hydroxyl groups. These results suggested that changes in the electrostatic or hydrogen-bonding ability of residues may contribute to the improved antibody binding to crude DNA. Interestingly, the two mutants with the best affinity, EP7 and EP27.1, obtained an 8-fold improvement in affinity after the corresponding mutations of EP27.1 Gly→Arg and EP7 Ile→His. The potential role of the amino acids Arg and His for binding DNA was highlighted in both mouse and human anti-DNA autoantibodies. Arg is the most important and variable amino acid in DNA binding. It can form hydrogen bonds with base-paired guanine, as well as unpaired and base-paired cytosines, and can fit into the major or minor DNA groove through extensive interactions with the DNA sugar phosphate backbone and with flexible side chains to promote binding. This may partially explain the improved affinity of EP27.1, EP2, and EP102, which have the same substitution with Arg. However, in one exceptional case, the substitution of Arg104 with Leu also increased the binding activity by 4-fold. Apparently, the simple accumulation of Arg substitutions does not increase the affinity of binding DNA. The modification of Arg 104→Leu may place Arg105 in a better position for interaction with DNA.

[0062] Analysis of the mutation sites on CDR3 of EP mutants derived from phage display and binding selection showed that residues 98-100, 105, 106, and 108 in H-CDR3 were the most conserved, and substitutions mainly occurred at residues 101-104, 107, and 109. In contrast, the highest frequency of mutations was observed at residues 101, 104, 106, and 109. The completely conserved residues may play a role in directly contacting the antigen and contribute more to determining specificity. We also found that the original H-CDR3 contains two Arg residues (Arg104, Arg105). This is consistent with anti-dsDNA autoantibodies having at least one, and in most cases two or three Args in H-CDR3. Arg104 may be substituted, and Arg105 is conserved. The conserved Arg105 was located in the center of H-CDR3, and was in a position that could effectively interact with DNA through hydrogen bonds and salt bridges. The conserved Arg105 combined with other conserved residues may play a critical role in DNA specific binding, and the residues with high mutation frequency may contribute more to modulate the affinity of the antibody.

[0063] The different order of reactivity intensity of the mutants in ELISA and immunofluorescence staining, and the different patterns of distribution in Raji cells, indicated that the epitopes of some mutants against the same antigen were changed or shifted due to the mutation of H-CDR3. This was tested using crude DNA for selection. The decision to use crude DNA for selection was based on the evidence that chTNT3 derived from Raji cell nuclei can bind mostly to single-stranded DNA. Although chTNT-3 showed a strong reaction to crude DNA, chTNT-3 may be more likely to bind to complex form DNA in the nucleus. EP / m51 showed approximately double the affinity of chTNT-3 to crude DNA and Raji cells, and similar in vivo biodistribution characteristics to chTNT-3. The tumor uptake % of EP / m51 also tended to be better than chTNT-3. All this suggested that humanization of mTNT-3 by CDR grafting combined with mutation was successful, and a humanized Ep antibody with improved affinity was obtained.

[0064] Therefore, it should be understood that (1) mutations on H-CDR3 are an efficient way to improve the affinity of humanized antibodies; (2) one humanized mutant (EP51) with the same specificity and higher affinity was obtained, which shows a good biodistribution profile required in vivo for clinical potential; (3) the results show that humanized antibodies against different epitopes of the same antigen can be obtained simultaneously, and the risk of specificity shift of humanized antibodies caused by CDR3 mutations can be avoided or controlled by selecting appropriate antigens; and (4) the information obtained by studying the specificity and affinity of EP mutants is useful for broadening the knowledge about chTNT3 binding to nuclear antigens and improving EP affinity and specificity. Finally, biodistribution studies in tumor-bearing mice showed that one candidate, EP35.2, had high and excellent absorption in tumors, good retention after 7 days of study, and appeared to be superior in vivo to chTNT-3 or fully human NHS76 in this experimental tumor model.

[0065] In some embodiments, numerical values ​​expressing quantities of ingredients, properties, such as concentrations, reaction conditions, and the like, used to describe and claim certain embodiments of the present invention should be understood to be modified in some cases by the term "about". Accordingly, in some embodiments, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending on the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as practicable. The numerical values ​​set forth in some embodiments of the present invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0066] The meanings of "a," "an," and "the" as used throughout the description herein and the claims which follow include plural references unless the context clearly dictates otherwise. The meaning of "in" as used in the description herein also includes "in" and "on," unless the context clearly dictates otherwise. Additionally, the term "coupled" as used herein is intended to include both direct coupling (where two elements are in contact with each other and coupled to each other) and indirect coupling (where at least one additional element is positioned between the two elements), unless the context clearly dictates otherwise. Thus, the terms "coupled" and "coupled with" are used interchangeably.

[0067] It should be apparent to those skilled in the art that many more modifications than those already described are possible without departing from the inventive concept herein. Thus, the subject matter of the present invention should not be limited, except as set forth in the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprise" and "comprising" should be interpreted as referring to an element, component, or step in a non-exclusive manner, indicating that the element, component, or step may be present, utilized, or combined with other elements, components, or steps not expressly mentioned. When the specification or claims refer to at least one of something selected from the group consisting of A, B, C... and N, the text should be interpreted as requiring only one element from that group, not A plus N, not B plus N, etc.

Claims

1. A humanized antinuclear antibody having improved binding affinity to a nuclear target compared to the corresponding non-humanized antibody.

2. The humanized antibody of claim 1 , wherein the nuclear target is single-stranded DNA.

3. The humanized antibody of claim 1 or 2, wherein the humanized antibody has a binding affinity at least two-fold higher than that of the corresponding non-human antibody.

4. The humanized antibody of any one of claims 1 to 3, wherein the humanized antibody has a binding affinity at least 4 times higher than the corresponding non-human antibody.

5. The humanized antibody of any one of claims 1 to 4, wherein the humanized antibody has a binding affinity at least 8 times higher than the corresponding non-human antibody.

6. The humanized antibody according to any one of claims 1 to 5, wherein the humanized antibody has mutated amino acids in H-CDR3 relative to the corresponding non-humanized antibody.

7. The mutated amino acids in H-CDR3 are １０７ Phe→Arg, １０１ Ile → His, １０２ Gly→Arg, １０９ Tyr→Thr, １０３ Val → Ser, １０４ Arg → Thr, １０９ Tyr→Arg, and １０４ The humanized antibody of claim 6 , wherein the amino acid sequence is selected from the group consisting of Arg→Leu.

8. The humanized antibody comprises: H-CDR1 sequence comprising TRYWMH (SEQ ID NO:1); H-CDR2 sequence comprising: GAIYPGNSDTSYNQKFKG (SEQ ID NO:2); H-CDR3 sequence comprising EEIGVRRWFAY (SEQ ID NO:3); L-CDR1 sequence comprising RARQSISNYLH (SEQ ID NO:4); L-CDR2 sequence comprising ASQSIS (SEQ ID NO:5); and L-CDR3 sequence including QQSNSWPLT (SEQ ID NO: 6) The humanized antibody according to any one of claims 1 to 7, having at least one of the following:

9. The humanized antibody comprises: 【Chemistry 1】 The humanized antibody of any one of claims 1 to 6, having a VH sequence comprising:

10. The humanized antibody comprises: 【Chemistry 2】 The humanized antibody of any one of claims 1 to 6, having a VL sequence comprising:

11. A hybrid molecule comprising at least a binding portion of an antibody according to any one of claims 1 to 10, which binds to at least one of a therapeutic agent and an imaging agent.

12. The at least binding portion of the antibody may be a full length antibody, a Fab fragment, a F(ab') ２ Fragment, Fab ２ 12. The hybrid molecule of claim 11, which is a fragment or scFv.

13. 13. The hybrid molecule of claim 11 or 12, wherein the therapeutic agent comprises at least one of a cytokine or a portion thereof, a chemokine or a portion thereof, an inhibitor of myeloid-derived suppressor cells (MDSC), an inhibitor of M2 macrophages, a radioisotope, a costimulatory molecule, a Toll-like receptor (TLR) agonist or ligand, a molecule that prevents epithelial-mesenchymal transition (EMT), and a chemotherapeutic drug.

14. 13. The hybrid molecule of claim 11 or 12, wherein the imaging agent comprises at least one of a radioisotope, a PET label, and a SPECT label.

15. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 10 or a hybrid molecule according to any one of claims 11 to 14.

16. 16. The pharmaceutical composition of claim 15, formulated for injection.

17. A method for targeting necrotic cells, comprising contacting said necrotic cells with an antibody according to any one of claims 1 to 10 or a hybrid molecule according to any one of claims 11 to 14.

18. The method of claim 17, wherein the necrotic cells are necrotic tumor cells within a tumor microenvironment.

19. 19. The method of claim 17 or 18, wherein the contacting step is carried out in vivo.

20. 1. A method for delivering a therapeutic agent to a tumor microenvironment containing necrotic tumor cells, comprising: Providing a therapeutic agent conjugated to an antibody according to any one of claims 1 to 10; contacting said necrotic tumor cells in said tumor microenvironment with said therapeutic agent under conditions in said tumor microenvironment that allow said antibody to bind to a nuclear target in said necrotic cells; The method includes:

21. 21. The method of claim 20, wherein the therapeutic agent comprises at least one of a cytokine or a portion thereof, a chemokine or a portion thereof, an inhibitor of myeloid-derived suppressor cells (MDSC), an inhibitor of M2 macrophages, a radioisotope, a costimulatory molecule, a Toll-like receptor (TLR) agonist or ligand, a molecule that prevents epithelial-mesenchymal transition (EMT), and a chemotherapeutic agent.

22. 22. The method of claim 20 or 21, wherein the tumor is a solid tumor.

23. The method of any one of claims 20 to 22, further comprising administering a vascular permeability enhancing agent.

24. 1. A method for delivering an imaging agent to a tumor microenvironment containing necrotic tumor cells, comprising: Providing an imaging agent conjugated to an antibody according to any one of claims 1 to 10; contacting said necrotic tumor cells in a tumor microenvironment with said imaging agent under conditions in which said antibody is capable of binding to a nuclear target in said necrotic cells within said tumor microenvironment; The method includes:

25. 25. The method of claim 24, wherein the imaging agent comprises at least one of a radioisotope, a PET label, and a SPECT label.

26. 26. The method of claim 24 or 25, wherein the tumor is a solid tumor.

27. The method of any one of claims 24 to 26, further comprising administering a vascular permeability enhancing agent.

28. 1. A method for generating an affinity-optimized humanized antibody derived from a murine antibody, comprising: identifying human antibody sequences that have a predetermined minimal identity with a murine antibody sequence; grafting the mouse CDRs for VH and VL into the VH and VL, respectively, of the identified human antibody sequences to obtain chimeric VH and VL sequences; generating scFv-hybrids using the chimeric VH and VL sequences and performing site-directed mutagenesis in the H-CDR3 to generate an scFv-hybrid library; identifying binders from said scFv-hybrid library using affinity selection; grafting the chimeric VH and VL sequences of the binder into the human antibody sequences to obtain the affinity-optimized humanized antibody. The method includes:

29. 29. The method of claim 28, wherein the predetermined minimum identity is at least 90% identity excluding the CDRs.

30. The method of claim 28 or 29, wherein the scFv-hybrid is a phage-displayed hybrid.

31. The method of any one of claims 28 to 30, wherein the site-directed mutagenesis in the H-CDR3 is directed to a single amino acid position in the H-CDR3.

32. The method of any one of claims 28 to 31, wherein the affinity selection is carried out over at least three successive selection / enrichment cycles.

33. The method according to any one of claims 28 to 32, wherein said affinity selection further comprises at least one step of additional site-directed mutagenesis in H-CDR3 and / or framework regions.

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