Methods and compositions comprising tagless blinatumomab

Abstract

Disclosed herein is a tagless bispecific two antibody (bsAb) biopharmaceutical with the hallmark ability to bind two separate targets. The two separate targets are T-cells and the proximal cancer cells to initiate an immune response. The disclosed bsAbs have extended half-lives with a focus on human serum albumin affibody (HSA)-fusions.

Description

[0001] METHODS AND COMPOSITIONS COMPRISING TAGLESS BLINATUMOMAB

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims benefit of U.S. Provisional Application No. 63 / 652,398, filed May 28, 2024, incorporated herein by reference in its entirety.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via PatentCenter encoded as XML in UTF-8 text. The electronic document, created on May 25, 2025, is entitled “103362-003W01_ST26.xml”, and is 41,443 bytes in size.

[0006] BACKGROUND

[0007] Approximately 15% of FDA-approved biotherapeutics are engineered to have half-life extending technology, with the most common method being PEGylation. There are several PEGylated therapeutic proteins currently approved. The first approved PEG-conjugated biotherapeutic is pegasparaginase (Oncaspar®), which was approved in 1984. Most PEGylated biotherapeutics were approved between 1999-2009, with only one, Pegloticase (Krystxxa®), being approved since then in 2014. PEGylation of therapeutic proteins not only helps slow renal clearance but is also cited to decrease proteolytic degradation (Gupta et al. 2019). Though generally considered safe, PEGylated therapeutic proteins have several known adverse events. One recent review even found that some patients had developed anti-PEG antibodies, despite the technology having historically thought to be non-immunogenic (Zhu et al. 2025). Further, there are some reports that PEGylation of certain proteins reduces its binding and activity (Muller et al. 2007).

[0008] Other half-life extending techniques include PASylation, direct Fc-fusion, direct human serum albumin-fusion (HSA) or indirect HSA association, and the addition of glycosylation sites (Kontermann 2016). The most significant factor of therapeutic protein clearance is the renal clearance size cut off, which is reported to be around 50-60 kDa. Surface charge also impacts clearance rates, where negatively charged proteins display slower levels of clearance compared to neutral or positively charged proteins (relative to the kidney physiology) (Kontermann 2016). A key mechanism of half-life extension is a protein’s ability to bind to neonatal Fc receptor (FcRn). The Fc region of human IgG and HSA bind to FcRn and provide increased half-life benefit. However, the two molecules bind to different targets on the receptor, therefore the rate of IgG (Fc) recycling is independent of that of HAS (and vice-versa) (Kontermann 2016). The interaction between the Fc domain on mAb therapeutics and the FcRn is the major reason for this class of drugs’ long half-lives.

[0009] Second generation bispecific antibodies (bsAbs) are currently being developed and approved with several of these half-life extending technologies in response to the short half-life problem of first generation bsAbs. Several Fc-fusion bsAbs are being investigated by bsAb leader Amgen, among other major pharmaceutical companies, start-ups, and academic institutions. Yet, there are some reasons why Fc-fused bsAbs may be undesirable despite its obvious advantages. Crosslinking of CD3 and the FcyR leads to nonspecific activation of immune cells which can result in cytokine release syndrome (CRS) (Wang et al. 2019; Borlak et al. 2016; Mandrup et al. 2021). Notably, clinical trials of Fc-fusion EpCAM-CD3 bispecific catumaxomab (Removab®) was recently terminated due to patient deaths following CRS among other adverse events credited to Fc-enhanced immune response (Borlak et al. 2016; Mandrup et al. 2021). This trial termination is a reminder than Fc-fusion bsAbs are not an immediate solution to the short half-life issue of bsAbs.

[0010] The first approved Fc-fusion therapeutic with an extended half-life was etanercept (Embrel®) produced by Amgen in 1998 for the treatment of rheumatoid arthritis and in some cases psoriasis (Lamanna et al. 2017). It is an anti-inflammatory tumor necrosis factor (TNF) inhibiting agent composed of dimers of the extracellular section of human TNFR2 fused to a human IgGl Fc. Etanercept has a half-life of 4 days, credited to the interaction of Fc with FcRn on human endothelial cells (Tracey et al. 2008; Ishino et al. 2013). This is a shorter half-life than full IgGl mAbs, which leads some researchers to believe that etanercept is subjected to steric hinderance (Tracey et al. 2007). A large review published in 2016 reports that despite some Fc- fusion biotherapeutics having comparable half-lives to full IgGs, they are generally regarded as unable to achieve the same half-lives as traditional mAbs. It is believed that some domains on the Fv regions of Fabs may contribute to interaction with FcRn, where association is favored in acidic conditions and disassociation is favored at natural conditions (Kontermann 2016).

[0011] Fc fusion proteins provide several benefits. Arguably the most significant benefit is the increase in a molecule’s half-life, but an increased hydrodynamic volume is another major factor. The Fc domain is ~36 kDa, which slows its renal clearance rate (SinoBio). An Fc-fusion is also promising for oncology therapies due to the presence of FcRn on some immune cells (Czajkowsky et al. 2012). From a protein engineering standpoint, the Fc domain folds independently and is stated to improve both solubility and stability. It also presents a major benefit for manufacturing as Fc-containing proteins can be purified via the highly optimized protein A platform (Czajkowsky et al. 2012).

[0012] In 2021, Amgen announced that they were working on a next-generation BiTE® with an extended half-life via Fc-fusion at The American Society of Hematology annual conference (Wu et al. 2015). More recent news has shared promising data on Tarlatamab, a half-life extended (HLE) BiTE for small-cell lung cancer. Phase 2 clinical trials published on the anti-CD3 anti- DLL3 bsAb reported strong positive results. The HLE bsAb is an Fc fusion which only needs to be administered once every two weeks (Ahn et al. 2023). The molecule exhibits half-life extension via fusion to the Fc domain (see U.S. Patent 7,635,472 and US Patent Application US20230235053A1, both herein incorporated by reference in their entirety).

[0013] Currently, the blinatumomab BiTE® used in treatment of CD 19-positive MRD(+) or R / R acute lymphoblastic leukemia (ALL) is given over an approximately one-month continuous infusion that requires patients to wear the medicine and pump in a backpack over the course of their treatment. Amgen’s Fc-fusion technology is reported to extend the serum half-life to 210 hours with positive in vitro and in vivo results for BiTEs® (Loren czewski et al. 2021). The abstract, published in Blood (2017), reports investigation into an anti-BCMA anti-CD3 BiTE® for multiple myeloma (MM) targeted at abnormal plasma cells in a patient’s bone marrow. While there are multiple treatments for MM, relapse is not uncommon in patients undergoing treatment

[0014] Human serum albumin (HSA) is a 67 kDa 585 amino acid globular protein that is the most abundant protein in human blood. It has a half-life of 19 days due to FcRn-mediated recycling and interaction with the Megalin / Cubilin complex, which helps to protect HSA from renal clearance (Muller et al. 2007; Kontermann 2016; Kwon et al. 2022; Pilati and Howard 2021). HSA is highly soluble and contains 17 disulfide bonds and one free thiol group.

[0015] HSA-related biotherapeutics can be separated into four major categories including HSA- drug nanoparticles, HSA-drug conjugates, HSA-binding pro-drugs, and HSA-based recombinant fusion proteins. Several HSA-associated oncology drugs have entered clinical trials following the approval of Abraxane (HSA-drug nanoparticle) in 2005.

[0016] Given the limitations described above, there is a need to explore new methods to determine and predict therapeutic outcomes using tagless bispecific antibodies in the treatment of cancer.

[0017] The methods disclosed herein address the need for potential clinical application in a context where bispecific antibodies are used as tumor immunotherapeutic other than antibodies exploring T cells potential in tumor microenvironment. SUMMARY

[0018] Disclosed are methods and compositions related to bispecific two single chains antibodies. Specifically, disclosed is a bispecific two single chain antibody comprising an amino acid sequence, wherein said amino acid sequence comprises SEQ ID NO: 1, wherein said amino acid sequence can comprise 1, 2, 3, 4, or 5 variations within its sequence, and wherein the sequence does not comprise any additional amino acids at either its N-terminus or its C-terminus.

[0019] Also disclosed is a bispecific two single chain antibody comprising an amino acid sequence, wherein said amino acid sequence comprises SEQ ID NO: 1, wherein said amino acid sequence can comprise 1, 2, 3, 4, or 5 variations within its sequence, and wherein the sequence does not comprise a tag at either its N-terminus or its C-terminus.

[0020] Further disclosed is an amino acid sequence consisting of SEQ ID NO: 1.

[0021] Disclosed is a method of making a bispecific two single chain antibody, the method comprising the steps of: a) providing an amino acid sequence comprising SEQ ID NO: 1, wherein said amino acid sequence can comprise 1, 2, 3, 4, or 5 variations within its sequence, and further wherein the amino acid sequence comprises a Cognate Binding Partner at one of its termini; b) exposing the amino acid sequence of step a) to an N-intein Ligand under conditions that the N- intein ligand and the Cognate Binding Partner form an intein complex; c) purifying the intein complex of step b); and d) exposing the intein complex to conditions which allow for the Cognate Binding Partner to be cleaved from the amino acid sequence comprising SEQ ID NO: 1 (wherein said amino acid sequence can comprise 1, 2, 3, 4, or 5 variations within its sequence); thereby making a bispecific two single chain antibody.

[0022] Also disclosed is a method of targeting CD3 site on T cells and CD 19 site on B cells, the method comprising exposing the cells to the bispecific two single chain antibody discussed above.

[0023] Further disclosed is a method of treating B cell-related conditions in a subject in thereof, the method comprising exposing the subject to the bispecific two single chain antibody discussed above.

[0024] Also disclosed herein is a kit comprising the bispecific two single chain antibody discussed above.

[0025] BRIEF DESCRIPTION OF FIGURES

[0026] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0027] FIG. 1 shows cleavage test of NpuC(+MF)-blinatumomab-his construct with anti-his primary antibody incubation overnight. FIG. 2 show Affibody molecule construct nucleotide sequence (SEQ ID NO: 1, SEQ ID NO: 27).

[0028] DETAILED DESCRIPTION

[0029] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiment s). To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof.

[0030] Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0031] Terminology

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. As used in this disclosure and in the appended claims, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise.

[0033] The following definitions are provided for the full understanding of terms used in this specification.

[0034] The terms “about” and “approximately” are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.

[0035] As used herein, the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation “may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

[0036] “Composition” refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “composition” is used, then, or when a particular composition is specifically identified, it is to be understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0037] The term “comprising”, and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed.

[0038] As used herein, the term “subject” or “individual” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0039] An “antibody” is a polypeptide that includes at least a light chain or heavy chain immunoglobulin variable region and specifically binds an epitope of an antigen. Antibodies include monoclonal antibodies, polyclonal antibodies, or fragments of antibodies. An antibody can be conjugated or otherwise labeled with a detectable label, such as an enzyme, hapten, or fluor ophore.

[0040] The term “standard therapy” or “conventional therapy” as used herein refers to ALL therapy using chemotherapy and / or HSCT. Acute lymphoblastic leukemia (ALL), including B- precursor acute lymphoblastic leukemia and other types of B (cell) lineage ALL, and treatments thereof are reviewed e.g. in Pui C H, Clin Adv Hematol Oneal. 4 (2006): 884-6; Pui C H, Evans WE, N Engl J Med 354 (2006): 166-178; Pui CH et al., Lancet 371 (2008): 1030-1043; Pui CH, Jeha S, Nat Rev Drug Discov 6 (2007): 149-165); Henze G, von Stackelberg A, Relapsed acute lymphoblastic leukemia. In: Childhood Leukemias, C-H Pui ed. Cambridge: Cambridge University Press; 2006, p. 473-486). Further information with respect to ALL can also be found e.g. under cancer.gov or leukemia-lymphoma.org.

[0041] The term “chemotherapy” as used herein denotes chemotherapy used for the treatment of acute lymphoblastic leukemia (ALL). Chemotherapy is the initial treatment of choice for ALL (see e.g. Pui et al. 2005). Most ALL patients end up receiving a combination of different treatments. In the treatment of ALL, there are no surgical options, due to the body-wide distribution of the malignant cells. In general, cytotoxic chemotherapy for ALL combines multiple anti-leukemic drugs in various combinations. Chemotherapy for ALL consists of three phases: remission induction, intensification, and maintenance therapy. Chemotherapy is also indicated to protect the central nervous system from leukemia. The aim of remission induction is to rapidly kill most tumor cells and get the patient into complete hematological remission. This is defined as the presence of less than 5% leukemic blasts in the bone marrow (as determined by light microscopy), For example, Clofarabine, Cyclophosphamide, VP16, Amsacrine, Prednisone, Melphalan, or Cytarabine, alone or in combination, is used to induce remission. Intensification uses high doses of intravenous multidrug chemotherapy to further reduce tumor burden. Typical intensification protocols use vincristine, cyclophosphamide, cytarabine, daunorubicin, etoposide, thioguanine or mercaptopurine given as blocks in different combinations. Since ALL cells sometimes penetrate the Central Nervous System (CNS), most protocols include delivery of chemotherapy into the CNS fluid commonly known as intrathecal chemotherapy. Some tumor centers deliver the drug through Ommaya reservoir (a device surgically placed under the scalp and used to deliver drugs to the CNS fluid and to extract CNS fluid for various tests). Other centers perform multiple lumbar punctures as needed for testing and treatment delivery. Intrathecal methotrexate or cytarabine is usually used for this purpose. The aim of maintenance therapy is to kill any residual cell that was not killed by remission induction, and intensification regimens. Although such cells are few, they will cause relapse if not eradicated. For this purpose, daily oral mercaptopurine, once weekly oral methotrexate, once monthly 5 -day course of intravenous vincristine an. The length of maintenance therapy is 3 years for boys, 2 years for girls and adults. Nervous system relapse is treated with intrathecal administration of hydrocortisone, methotrexate, and cytarabine. As the chemotherapy regimens can be intensive and protracted (often about 2 years in case of the GMALL UKALL, HyperCVAD or CALGB protocols; about 3 years for males on COG protocols), many patients have an intravenous catheter inserted into a large vein (termed a central venous catheter or a Hickman line), or a Portacath (a cone-shaped port with a silicone nose that is surgically planted under the skin, usually near the collar bone).

[0042] The term “bispecific single chain antibody” or “single chain bispecific antibody” or related terms in accordance with the present invention mean antibody constructs resulting from joining at least two antibody single chain variable regions in a single polypeptide chain devoid of the constant and / or Fe portion(s) present in full immunoglobulins. The bispecific two single chain antibody as referred to herein is functional, i.e. cytotoxically active, as a monomer and therefore clearly distinguishable from diabodies described in the art which are functional only as dimers or multimers.

[0043] Bispecific antibodies comprising specificities for human CD 19 and human CD3 which are not of the single-chain format and which retarget T-cell cytotoxicity to lymphoma cells in an MHC-independent manner have already been shown to be effective in vivo in animal models (Bohlenl992) as well as in some pilot clinical trials. So far these antibodies were constructed by hybrid-hybridoma techniques, by covalently linking the monoclonal antibodies (Anderson, Blood 80 (1992), 2826-34) or by a diabody approach (Kipriyanov, Int. J. Cancer 77 (1998), 763-772). More extensive clinical studies have been hampered by the fact that these antibodies have low biological activity such that low dosages have to be administered and that application of the antibodies alone did not provide for a beneficial therapeutic effect. Furthermore, the availability of clinical grade material was limited. The prior art has exemplified bispecific two single chain antibodies comprising specificities for both human CD3 and human CD 19 antigens (Loftier, Blood 95 (2000), 2098-103; WO99 / 54440; Dreier, Int. J. Cancer. 100 (2002), 690-7). WO 99 / 54440 documents the successful clinical use of a construct in the format V, (CD19)-V (CD19)- V(CD3)-V, (CD3) and stresses that the order of variable domains within the construct is not decisive. Yet, in particular for distinct clinical and pharmaceutical uses, constructs have to be provided which can be produced in large amounts by reasonably high levels of expression of the recombinant constructs and by adequate purification methods after expression. In the event that extremely low amounts of pure protein are obtained, it becomes prohibitively cumbersome and / or costly to generate therapeutically relevant amounts of Such constructs. In the special case of proteinaceous medicaments intended for parental administration, these medicaments should be highly active and potent, even in low concentrations, in order to avoid adverse side-effects due to excessive protein concentrations or Voluminous infusion / inj ection Solutions. Disadvantages of highly dosed proteinaceous medicaments or highly-dosed medicaments based on nucleic acids comprise, inter alia, the promotion of hypersensitivities and inflammatory events, in particular at the site of administration. Thus, the technical problem of the present invention is the provision of means and methods for the generation of well tolerated and convenient medicaments for the treatment and or amelioration of B-cell related or B-cell mediated disorders. Accordingly, the present invention relates to a pharmaceutical composition comprising a bispecific two single chain antibody construct, said bispecific two single chain antibody construct comprising binding domains specific for human CD3 and human CD 19. The corresponding variable heavy chain region (VH) and corresponding variable light chain region (VL) region in said CD 19 x CD3 bispecific single chain antibody construct can be, from the N-terminal to the C-terminal, VL (CD19) - VH(CD19) - VH(CD3) -VL (CD3), arranged in this order.

[0044] A “linker” as used herein connects V domains of the same specificity, whereas a “spacer” as used herein connects V domains of different specificities. For example, a bispecific two single chain antibody may be a construct with a total of two antibody variable regions, for example two VH regions, each capable of specifically binding to a separate antigen, and connected with one another through a short (usually less than 10 amino acids) synthetic polypeptide spacer such that the two antibody variable regions with their interposed spacer exist as a single contiguous polypeptide chain. Another example of a bispecific two single chain antibody may be a single polypeptide chain with three antibody variable regions. Here, two antibody variable regions, for example one VH and one VL, may make up an scFv, wherein the two antibody variable regions are connected to one another via a synthetic polypeptide linker, the latter often being genetically engineered so as to be minimally immunogenic while remaining maximally resistant to proteolysis. This scFv is capable of specifically binding to a particular antigen, and is connected to a further antibody variable region, for example a VH region, capable of binding to a different antigen than that bound by the scFv. Yet another example of a bispecific single chain antibody may be a single polypeptide chain with four antibody variable regions. Here, the first two antibody variable regions, for example a VH region and a VL region, may form one scFv capable of binding to one antigen, whereas the second VH region and VL region may form a second scFv capable of binding to another antigen. Within a single contiguous polypeptide chain, individual antibody variable regions of one specificity may advantageously be separated by a synthetic polypeptide linker as described above, whereas the respective scFvs may advantageously be separated by a short polypeptide spacer as described above. Non-limiting examples of bi specific two single chain antibodies as well as methods for producing them are shown in WO 99 / 54440, WO 2004 / 106381, WO 2007 / 068354, Mack, J. Immunol. (1997), 158, 3965-70; Mack, PNAS, (1995), 92, 7021-5; Kufer, Cancer Immunol. Immunother., (1997), 45, 193-7; Loftier, Blood, (2000), 95, 6, 2098-103; Briihl, J. Immunol., (2001), 166, 2420-2426.

[0045] The term “allogeneic hematopoietic stem cell transplantation (HSCT)” as used herein means allogeneic hematopoietic stem cell transplantation (HSCT) or bone marrow transplantation which is a medical procedure in the field of hematology and oncology that involves transplantation of hematopoietic stem cells (HSC). It is most often performed for patients with diseases of the blood or bone marrow, or certain types of cancer, such as ALL. Most recipients of HSCTs are leukemia (e.g. ALL) patients who would benefit from treatment with high doses of chemo therapy or total body irradiation. Allogeneic HSCT in children with ALL is described e.g. in Schrauder A, et al. (Bone Marrow Transplantation 41 (2008): Suppl2 S71-74).

[0046] The term “minimal residual disease (MRD)” as defined herein denotes a term used after treatment e.g. with chemotherapeutics when leukemic cells cannot be found in the bone marrow using standard tests, such as microscopic methods. Rather, more sensitive tests such as flow cytometry (FACS based methods) or polymerase chain reaction (PCR) have to be used in order to find evidence that leukemia cells remained in the bone marrow of the ALL patient. More specifically, the presence of leukemia cells below the cytological detection limit (5% leukemic cells) is defined as minimal residual disease (MRD). If no MRD is detectable (<10‘4, i.e. less than 1 leukemia cell per 104bone marrow cells detectable), a complete molecular remission is reached (MRD negativity or MRD negative status). An “MRD positive status” as defined herein means a signal measured by PCR or FACS above detection limit or a quantitative threshold. An “MRD negative status” as defined herein means below detection limit and / or below a quantitative threshold measured by PCR or FACS. The prognostic value of minimal residual disease quantification in childhood ALL has been described e.g. in Bader et al. (J. Clin. Oneal. 27 (2009): 377-384) or Eckert et al. (Lancet 358 (2001): 1239-41). The MRD status can be measured by PCR or FACS analysis in that the individual cytogenetic abnormalities mentioned herein, and / or rearrangements of immunoglobulin genes or T-cell receptor (TCR) rearrangements are quantitatively detected. For example, PCR analysis can detect fusion transcripts such as bcr / abl or t(4;l 1) translocations as well as individual clonal rearrangements of immunoglobulins (IgH) and / or T-cell receptor genes (TCR).

[0047] The term “molecular relapse” as used herein means that said patient shows a signal for a cytogenetic abnormality above detection limit and / or at least one marker by rearrangement with a sensitivity of >10'4by PCR and / or FACS, as set forth above. In another preferred embodiment of the pharmaceutical methods and means of the invention, the corresponding variable heavy chain regions (VH) and the corresponding variable light chain regions (VL) regions in said CD19xCD3 bispecific two single chain antibody constructs are arranged, from N-terminus to C-terminus, in the order, VL(CD19)-VH, (CD19)-VH (CD3)- VL(CD3).

[0048] The term “remission” or “complete hematological remission” as used herein is to be understood as having no evidence of ALL disease after standard treatment, e.g. after chemotherapy and / or transplantation. This means that the bone marrow contains fewer than 5% blast cells as determined by light microscopy, the blood cell counts are within normal limits, and there are no signs or symptoms of the ALL disease. Nevertheless, it may occur that not all leukemia cells could be eliminated from the body. Such a patient, though staged as being in remission or complete hematological remission, is still MRD positive. These remaining tumor cells may give rise to recurrent leukemia. The pharmaceutical means and methods of the invention can be used to kill these remaining tumor cells in order to prevent recurrence of the leukemia originating from the occult leukemia cells remaining in the body after primary therapy. In this way, the pharmaceutical means and methods help to prevent disease relapse in ALL patients.

[0049] In contrast, a “molecular complete remission” means that there is no evidence of leukemia cells in biopsies of the bone marrow, even when using very sensitive tests such as PCR or FACS analysis. Put in other words: If no MRD is detectable (<10‘4, i.e. less than one leukemia cell per 104bone marrow cells), a complete molecular remission is reached.

[0050] In another preferred embodiment of the pharmaceutical methods and means of the invention, the time to molecular relapse (detectable by the assays described above) is more than 6 months, preferably more than 7, 8, 9, 10, 11 or 12 months, or even more preferred for 2, 3, 4, 5 or more years.

[0051] The uninterrupted administration of the CD19x specific single chain antibody may be intravenous, CD3 bi-parenteral, subcutaneous, transdermal, intraperitoneal, intra- muscular or pulmonary. The intravenous mode of administration will in most cases be the mode of choice for uninterruptedly administering the CD19xCD3 bispecific two single chain antibody and, as the case may be, for coadministration of a pharmaceutical agent as part of a regimen of co therapy. As such, intravenous administration is especially preferred. In this case, a suitable metering device such as the multi-therapy infusion pump model 6060 manufactured by Baxter may advantageously be chosen. Whatever metering device is chosen, it should be of such design and construction as to minimize or, better, preclude an interruption of administration of therapeutic agent in the event of cartridge exchange and / or power cell replacement or recharging. This may be accomplished, for example by choosing a device with a secondary reservoir of CD19xCD3 bispecific two single chain antibody solution apart from the cartridge to be exchanged so that continuous infusion from this secondary reservoir into the patient may continue even while the empty or almost empty cartridge is removed and replaced with a fresh one.

[0052] In a most preferred embodiment, the invention relates to the use of a bispecific two single chain antibody construct, a nucleic acid sequence, a vector and / or a host as defined herein for the preparation of a pharmaceutical composition for the prevention, treatment or amelioration of a proliferative dis- ease, a minimal residual cancer, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, allergic reactions, parasitic reactions, graft-versus-host diseases host- versus-graft diseases or B-cell malignancies, wherein said pharmaceutical composition optionally further comprises a proteinaceous compound capable of providing an activation signal for immune effector cells. Accordingly, a method for the prevention, treatment or amelioration of a proliferative disease, a minimal residual cancer, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, viral disease, allergic reactions, parasitic reactions, graft-versus-host diseases, host-versus-graft diseases, or B-cell malignancies is provided, whereby said method comprises the step of administering to a subject in need of such a prevention, treatment or amelioration a pharmaceutical composition of the invention. Most preferably said subject is a human. The disease to be treated with the pharmaceutical composition of the invention may be a minimal residual cancer, for example, a minimal residual lymphoma or leukemia. The autoimmune disease to be treated with the pharmaceutical composition of the invention may be in inflammatory autoimmune disease, for example, rheumatoid arthritis.

[0053] In accordance with this invention, it is also envisaged that a bispecific two single chain antibody construct, a nucleic acid sequence, a vector and / or a host as described herein is / are used for the preparation of a pharmaceutical composition for depletion of B-cells. The B cell malignancy to be treated with the pharmaceutical composition of the invention is in a most preferred embodiment non-Hodgkin lymphoma, B-cell leukemias or Hodgkin lymphoma.

[0054] Accordingly, the present invention provides for a method for the treatment of B-cell malignancies, B-cell mediated autoimmune diseases or the depletion of B-cells and / or for a method delaying a pathological condition which is caused by B-cell disorders comprising administering the pharmaceutical composition of the invention into a mam- mal, preferably a human, affected by said malignancies, disease and / or pathological condition. The invention provides for a kit comprising a bispecific two single chain antibody construct, a nucleic acid sequence, a vector and / or a host as defined above. Said kit is particularly useful in the preparation of the pharmaceutical composition of the present invention and may, inter alia, consist of a container useful for injections or infusions. Advantageously, the kit of the present invention further comprises, optionally (a) buffer(s), storage solutions and / or remaining reagents or materials required for the conduct of medical or scientific purposes. Furthermore, parts of the kit of the invention can be packaged individually in vials or bottles or in combination in containers or multi-container units. The kit of the present invention may be advantageously used, inter alia, for carrying out the method of the invention and could be employed in a variety of applications referred herein, e.g., as research tools or medical tools. The manufacture of the kits preferably follows standard procedures which are known to the person skilled in the art.

[0055] The term “contacting” as used herein refers to bringing two biological entities together in such a manner that the compound can affect the activity of the target, either directly; i.e., by interacting with the target itself, or indirectly; i.e., by interacting with another molecule, co-factor, factor, or protein on which the activity of the target is dependent. “Contacting” can also mean facilitating the interaction of two biological entities, such as peptides, to bond covalently or otherwise.

[0056] As used herein, the terms “target protein,” “protein of interest,” and “therapeutic agent” include any synthetic or naturally occurring protein or peptide. In the context of this invention, a “protein of interest” is a bispecific two single chain antibody, such as those found in SEQ ID NO: 1 or variants thereof. It is to be purified using split intein purification technology by an end user in a laboratory or manufacturing setting, as opposed to any context related to the manufacture of the purification medium itself.

[0057] As used herein, “variant” refers to a molecule that retains a functional activity that is the same or substantially similar to that of the original sequence. The variant may be from the same or different species or be a synthetic sequence based on a natural or prior molecule. Moreover, as used herein, “variant” refers to a molecule having a structure attained from the structure of a parent molecule (e.g., a protein or peptide disclosed herein) and whose structure or sequence is sufficiently similar to those disclosed herein that based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities compared to the parent molecule. For example, substituting specific amino acids in a given peptide can yield a variant peptide with similar activity to the parent. Specifically, as it pertains to SEQ ID NO: 1, a variant can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes within SEQ ID NO: 1, as long as SEQ ID NO: 1 is able to retain its ability to act as a bispecific two single chain antibody and bind CD3 and CD 19 as described herein. It is further noted that the “variant” described herein does not comprise a His Tag or a Flag Tag. In a specific example, the composition does not comprise any additional amino acid residues on either the N-terminus or C-terminus. As used herein, the term “amino acid sequence” refers to a list of abbreviations, letters, characters or words representing amino acid residues. The amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; C, cysteine; D aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine.

[0058] “Peptide” as used herein refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A peptide is comprised of consecutive amino acids. The term “peptide” encompasses naturally occurring or synthetic molecules.

[0059] In addition, as used herein, the term “peptide” refers to amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, etc. and may contain modified amino acids other than the 20 gene-encoded amino acids. The peptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in the peptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification can be present in the same or varying degrees at several sites in a given polypeptide. Also, a given peptide can have many types of modifications. Modifications include, without limitation, linkage of distinct domains or motifs, acetylation, acylation, ADP-ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, sei enoyl ati on, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation. (See Proteins — Structure and Molecular Properties 2nd Ed., T. E. Creighton, W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983)).

[0060] As used herein, “isolated peptide” or “purified peptide” is meant to mean a peptide (or a fragment thereof) that is substantially free from the materials with which the peptide is normally associated in nature, or from the materials with which the peptide is associated in an artificial expression or production system, including but not limited to an expression host cell lysate, growth medium components, buffer components, cell culture supernatant, or components of a synthetic in vitro translation system. The peptides disclosed herein, or fragments thereof, can be obtained, for example, by expression of a recombinant nucleic acid encoding the peptide (for example, in a cell or in a cell-free translation system), or by chemically synthesizing the peptide. In addition, peptide fragments may be obtained by any of these methods, or by cleaving full length proteins and / or peptides.

[0061] The word “or” as used herein means any one member of a particular list and also includes any combination of members of that list.

[0062] The phrase “nucleic acid” as used herein refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense, which is capable of hybridization to a complementary nucleic acid by Watson-Crick base-pairing. Nucleic acids of the invention can also include nucleotide analogs (e.g., BrdU), and non -phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combination thereof.

[0063] As used herein, “isolated nucleic acid” or “purified nucleic acid” is meant to mean DNA that is free of the genes that, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, such as an autonomously replicating plasmid or virus; or incorporated into the genomic DNA of a prokaryote or eukaryote (e.g., a transgene); or which exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR, restriction endonuclease digestion, or chemical or in vitro synthesis). It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequences. The term “isolated nucleic acid” also refers to RNA, e.g., an mRNA molecule that is encoded by an isolated DNA molecule, or that is chemically synthesized, or that is separated or substantially free from at least some cellular components, for example, other types of RNA molecules or peptide molecules.

[0064] “Intein” refers to an in-frame intervening sequence in a protein as described by Perler (Perler, Davis et al. 1994). An intein can catalyze its own excision from the protein through a post- translational protein splicing process to yield the free intein and a mature protein. An intein can also catalyze the cleavage of the intein-extein bond at either the intein N-terminus, or the intein C-terminus, or both of the intein-extein termini. As used herein, “intein” encompasses mini- inteins, modified or mutated inteins, and split inteins.

[0065] The term “Split Intein” refers to a pair of two distinct and separately translated protein segments, comprising an “N-Terminal Intein Segment” (INTN) and a counterpart “C-Terminal Intein Segment” (INTc) binding partner, which are characterized by at least one of the following properties:

[0066] (1) INTN and INTc segments exhibit an innate affinity for their respective counterpart protein, which drive the pair to spontaneously associate, fold, and non-covalently “bind” together, forming an “Intein Complex”.

[0067] (2) Upon association, an Intein Complex may become “Splicing Active” or “Cleaving Active”, wherein the complex catalyzes cleaving or splicing events between the complex and its extein fusion partners. This activity is generally considered to be contingent upon formation of the Intein Complex, which is to say that neither INTN nor INTc possess said activity autonomously in the absence of their binding partner.

[0068] (3) INTN and INTc segments containing peptides, protein domains, or amino acid sequences that are identical, similar to, or derived from naturally occurring or artificially split inteins, such as those cataloged in the so-called “InBase, The Intein Database” established by Perl er (Perl er 1999, Perler 2002).

[0069] (4) It should be noted though that the formation of complexes exhibiting cleaving and / or splicing activity is not strictly required to satisfy the definition of “Split Intein” and / or INTN and / or INTc segments. In other words, for example, if a “Split Intein” has been modified so that it no longer possesses the characteristic of exhibiting splicing and / or cleaving activity, it is still encompassed by this invention.

[0070] The term “Cognate Binding Partner” or “Cognate” refers to any peptide or protein segment capable of spontaneous, non-covalent association with any “Binding Active” INTN counterpart it contacts. Cognate Binding Partners include, but are not limited to, the subset of peptides and protein segments that comprise species defined as INTc peptides, including INTc peptides that have been operably linked to additional linker and tag moieties. For example, an INTc segment may be an example of a Cognate Binding Partner, but a Cognate Binding Partner is not by definition strictly required to be a species of INTc.

[0071] INTc are also herein further differentiated from the Cognate superfamily in that INTc are specifically those Binding Partners that associate with INTN to form an ACTIVE Intein Complex. INTc should be considered a Cognate if it associates with INTN and folds into an Intein Complex, but the resulting complex is an INACTIVE Intein Complex (exhibits no splicing or cleaving activity). “Cognate Binding Partner” can exclude tags, as defined below.

[0072] As used herein, the term “extein” refers to any peptide, protein, domain, or amino acid that is expressed covalently in fusion to either the N-terminus of an INTN segment, the C-terminus of an INTc segment. Exteins are further characterized as the portion of said intein-fused polypeptide which may be cleaved or spliced upon excision of the intein or intein complex.

[0073] The N-terminal Extein (N-EXT) is specifically the Extein expressed in fusion with the N- terminus of the INTN segment. An N-EXT is only classified as such if expressed in fusion with an INTN segment, however, an INTN segment does not strictly require the presence of an N-EXT to satisfy the definition of I TN segment.

[0074] The C-terminal Extein (C-EXT) is specifically the Extein expressed in fusion with the C- terminus of an INTc segment or cognate binding partner. A C-EXT is only classified as such if expressed in fusion with an INTc segment or cognate binding partner, however, INTc segments and cognate binding partners do not strictly require the presence of a C-EXT to satisfy their respective definitions.

[0075] Furthermore, N-EXT and C-EXT domains may continue to be identified as such after cleaving or splicing events occur, despite being excised from their respective INTN and INTc fusion partners.

[0076] The term “N-Intein Ligand” refers to a protein that has been (or will be) immobilized onto a solid surface, substrate or chromatographic medium to function as an affinity ligand. As defined herein, the N-Intein Ligand is comprised of an INTN segment at minimum but may also be comprised of additional operably linked proteins, peptides, functional domains, amino acid motifs and or chemical moieties, which are expressed as fusion partners with the INTN segment. Fusion partners that comprise the N-Intein Ligand may include (but are not limited to) a Sensitivity Enhancing Motif (SEM), as well as various “Immobilization Moieties”, “Linker Moieties”, and / or “Tag Moieties”, which collectively are referred to as “ILT Moieties”.

[0077] The term “Sensitivity Enhancing Motif’ (SEM) refers to an amino acid sequence of three or more residues expressed in fusion with the N-terminus of an INTN segment, which renders the splicing or cleaving activity of an intein complex highly sensitive to extrinsic conditions as described previously in U.S. Patent 10,066,027. The SEM is a constitutive element of an N-Intein Ligand but is distinct from the INTN segment and other fusion partners that may comprise said N- Intein Ligand.

[0078] “ILT Moieties” is a collective term for one or more amino acids expressed as fusion partners with an INTN to comprise an N-Intein Ligand. ILT moieties can be further subdivided into constituent groups that include at least one of the “immobilization” (I), “linker” (L), and / or “tag” (T) moiety classifications that are defined further below, individual moieties are operably linked, and may be trivially repeated, combined or rearranged in relation to each other, and in relation to the INTN. The term “immobilization moiety” refers to one or more amino acid residues (e.g. Cys), expressed in fusion with the INTN, which allows for covalent immobilization of the N-Intein Ligand (and its fusion partners by extension).

[0079] The classification “linker moiety” or “linker” refers to one or more amino acid residues expressed in fusion with the INTN that confers structure, spacing, or flexibility between the I TN, the immobilization moiety, and / or other fusion partners. Common examples of linker moieties include, but are not limited to: Glycine- Serine repeat ((GlyniSern2) ns), Polyproline dyad ((XaaPro)n), and a-helical (A(EAAAK)nA) linker motifs.

[0080] The classification “tag moiety” or “tag” refers to a peptide, domain, or a specific amino acid motif that is expressed in fusion with a protein, and aids in purification, detection, and / or enhances soluble expression of its fusion partners. Examples of common “tag” moieties include but are not limited to: purification tags (e.g. poly-His, poly-Arg, GST, CBD, MBP, CBP, Strep- Tag, FLAG-tag, etc.), detection tags (e.g. GFP, luciferase, epitope tags (i.e. FLAG, HA, c-myc), HRP, etc.), and express! on / solubility enhancing tags (e.g. T7-tag, NusA, TrxA, DsbA, DsbC, GST, MBP, etc.). In some embodiments, these tags are specifically excluded for use with the bispecific two single chain antibodies disclosed herein.

[0081] An INTN, INTC or Cognate Binding Partner domain is considered “Binding Active” if the segment exhibits affinity for its counterpart binding partner and can participate in a Binding Event that forms a new Intein Complex. The terms “Binding Active” and “Binding Inactive” are used to distinguish functional, singular INTN, INTC and / or Cognate segments from otherwise compositionally identical segments, which have (a) already bound a partner to form an Intein Complex, or (b) misfolded in such a way as to suppress the segment’s affinity for its potential binding partners. Importantly, when comprising an Intein Complex, constituent INTN, INTC and / or Cognate segments can bind each other such that they cannot further associate with additional otherwise compatible binding partners that they might encounter while the Intein Complex exists. For example, a given INTN and INTc may associate and bind each to form an Intein Complex, but upon formation of said complex, the INTN and INTc can become functionally “Binding Inactive” - neither segment can participate in any further binding events while comprising the Intein Complex. However, if the Intein Complex is dissolved, and the INTN and INTc are dissociated and subsequently refolded such that their affinity is restored, the individual segments may again become “Binding Active”.

[0082] An Intein Complex can be further functionally classified as either “INACTIVE” or “ACTIVE” with respect to intein splicing and / or cleaving activity. An INACTIVE Intein Complex is one where the Intein Complex exhibits less than 10% cleaving or splicing behavior with its Extein fusion partners. Conversely, An ACTIVE Intein Complex is one where the catalyzes a cleaving or splicing event that alters the peptide bonds of at least one of its Extein fusion partners. An ACTIVE Intein Complex may be further categorized by the specific type of canonical intein event that it catalyzes: C-Terminal Cleaving, N-Terminal Cleaving, Dual Cleaving, or Splicing. Once an “Active Intein Complex” catalyzes a cleaving or splicing event, the resulting Intein Complex may have no further effect on the peptide bonds of its fusion partners (splicing and cleaving reactions are irreversible), and thus the resulting Intein Complex can generally be considered an “INACTIVE Intein Complex” after catalyzing any cleaving or splicing event. By “no further effect” is meant less than a 10% effect.

[0083] As used herein, the term “splice” or “splices” means to excise a central portion of a polypeptide to form two or more smaller polypeptide molecules. In some cases, splicing also includes the step of fusing together two or more of the smaller polypeptides to form a new polypeptide. Splicing can also refer to the joining of two polypeptides encoded on two separate gene products through the action of a split intein.

[0084] As used herein, the terms “cleave”, “cleaves”, “cleavage” and “a cleaving event” refer to a chemical reaction in which a peptide bond within a polypeptide is broken, thereby dividing a single polypeptide to form two or more smaller polypeptide molecules. In some cases, cleavage is mediated by the addition of an extrinsic endopeptidase, which is often referred to as “proteolytic cleavage”. In other cases, cleaving can be mediated by the intrinsic activity of one or both of the cleaved peptide sequences, which is often referred to as “self-cleavage”. Cleavage can be controlled by extrinsic conditions (such as buffer pH), as in the action of the split intein system described herein.

[0085] By the term “fused” or “in fusion with” is meant covalently bonded to. For example, a first peptide is fused to a second peptide when the two peptides are covalently bonded to each other (e.g., via a peptide bond). Peptides and / or protein domains conjoined by peptide bonds may also be referred to as “fusion partners”.

[0086] As used herein an “isolated” or “substantially pure” substance is one that has been separated from components which naturally accompany it. Typically, a polypeptide is substantially pure when it is at least 50% (e.g., 60%, 70%, 80%, 90%, 95%, and 99%) by weight free from the other proteins and naturally-occurring organic molecules with which it is naturally associated.

[0087] Herein, “bind”, “binds”, “binding” or “binding event” means that one molecule recognizes and adheres to another molecule in a sample, but does not substantially recognize or adhere to other molecules in the sample. The terms “bind”, “binds”, “binding” and “binding event” also imply the interaction between two molecules is non-covalent and reversible. One molecule “specifically binds” another molecule if it has a binding affinity greater than about 105to 106liters / mole for the other molecule. These terms are used interchangeably with “associate with,” “associates with,” or “associating with.”

[0088] Nucleic acids, nucleotide sequences, proteins or amino acid sequences referred to herein can be isolated, purified, synthesized chemically, or produced through recombinant DNA technology. All of these methods are well known in the art.

[0089] As used herein, the terms “modified” or “mutated,” as in “modified intein” or “mutated intein,” refer to one or more modifications in either the nucleic acid or amino acid sequence being referred to, such as an intein, when compared to the native, or naturally occurring structure. Such modification can be a substitution, addition, or deletion. The modification can occur in one or more amino acid residues or one or more nucleotides of the structure being referred to, such as an intein.

[0090] As used herein, “operably linked” refers to the association of two or more biomolecules in a configuration relative to one another such that the normal function of the biomolecules can be performed. In relation to nucleotide sequences, “operably linked” refers to the association of two or more nucleic acid sequences, by means of enzymatic ligation or otherwise, in a configuration relative to one another such that the normal function of the sequences can be performed. For example, the nucleotide sequence encoding a pre-sequence or secretory leader is operably linked to a nucleotide sequence for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence; and a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation of the sequence.

[0091] “Sequence homology” can refer to the situation where nucleic acid or protein sequences are similar because they have a common evolutionary origin. “Sequence homology” can indicate that sequences are very similar. Sequence similarity is observable; homology can be based on the observation. “Very similar” can mean at least 70% identity, homology or similarity; at least 75% identity, homology or similarity; at least 80% identity, homology or similarity; at least 85% identity, homology or similarity; at least 90% identity, homology or similarity; such as at least 93% or at least 95% or even at least 97% identity, homology or similarity. The nucleotide sequence similarity or homology or identity can be determined using the “Align” program of Myers et al. (1988) CAB IOS 4: 11-17 and available at NCBI. Additionally or alternatively, amino acid sequence similarity or identity or homology can be determined using the BlastP program (Altschul et al. Nucl. Acids Res. 25:3389-3402), and available at NCBI. Alternatively or additionally, the terms “similarity” or “identity” or “homology,” for instance, with respect to a nucleotide sequence, are intended to indicate a quantitative measure of homology between two sequences.

[0092] Alternatively or additionally, “similarity” with respect to sequences refers to the number of positions with identical nucleotides divided by the number of nucleotides in the shorter of the two sequences wherein alignment of the two sequences can be determined in accordance with the Wilbur and Lipman algorithm. (1983) Proc. Natl. Acad. Sci. USA 80:726. For example, using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4, and computer-assisted analysis and interpretation of the sequence data including alignment can be conveniently performed using commercially available programs (e.g., Intelligenetics™ Suite, Intelligenetics Inc. CA). When RNA sequences are said to be similar or have a degree of sequence identity with DNA sequences, thymidine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence. The following references also provide algorithms for comparing the relative identity or homology or similarity of amino acid residues of two proteins, and additionally or alternatively with respect to the foregoing, the references can be used for determining percent homology or identity or similarity. Needleman et al. (1970) J. Mol. Biol. 48:444-453; Smith et al. (1983) Advances App. Math. 2:482-489; Smith et al. (1981) Nuc. Acids Res. 11 :2205-2220; Feng et al. (1987) J. Molec. Evol. 25:351-360; Higgins et al. (1989) CABIOS 5: 151-153; Thompson et al. (1994) Nuc. Acids Res. 22:4673-480; and Devereux et al. (1984) 12:387-395. “Stringent hybridization conditions” is a term which is well known in the art; see, for example, Sambrook, “Molecular Cloning, A Laboratory Manual” second ed., CSH Press, Cold Spring Harbor, 1989; “Nucleic Acid Hybridization, A Practical Approach”, Hames and Higgins eds., IRL Press, Oxford, 1985; see also FIG. 2 and description thereof herein wherein there is a sequence comparison.

[0093] The terms “plasmid” and “vector” and “cassette” refer to an extrachromosomal element often carrying genes which are not part of the central metabolism of the cell and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell. Typically, a “vector” is a modified plasmid that contains additional multiple insertion sites for cloning and an “expression cassette” that contains a DNA sequence for a selected gene product (i.e., a transgene) for expression in the host cell. This “expression cassette” typically includes a 5' promoter region, the transgene ORF, and a 3' terminator region, with all necessary regulatory sequences required for transcription and translation of the ORF. Thus, integration of the expression cassette into the host permits expression of the transgene ORF in the cassette.

[0094] The term “buffer” or “buffered solution” refers to solutions which resist changes in pH by the action of its conjugate acid-base range.

[0095] The term “loading buffer” or “binding buffer” refers to the buffer containing the salt or salts which is mixed with the protein preparation for loading the protein preparation onto a column. This buffer is also used to equilibrate the column before loading, and to wash to column after loading the protein.

[0096] The term “wash buffer” is used herein to refer to the buffer that is passed over a column (for example) following loading of a protein of interest (such as one coupled to a C-terminal intein fragment, for example) and prior to elution of the protein of interest. The wash buffer may serve to remove one or more contaminants without substantial elution of the desired protein.

[0097] The term “elution buffer” refers to the buffer used to elute the desired protein from the column. As used herein, the term “solution” refers to either a buffered or a non-buffered solution, including water.

[0098] The term “washing” means passing an appropriate buffer through or over a solid support, such as a chromatographic resin.

[0099] The term “eluting” a molecule (e.g. a desired protein or contaminant) from a solid support means removing the molecule from such material.

[0100] The term “contaminant” or “impurity” refers to any foreign or objectionable molecule, particularly a biological macromolecule such as a DNA, an RNA, or a protein, other than the protein being purified, that is present in a sample of a protein being purified. Contaminants include, for example, other proteins from cells that express and / or secrete the protein being purified.

[0101] The term “separate” or “isolate” as used in connection with protein purification refers to the separation of a desired protein from a second protein or other contaminant or mixture of impurities in a mixture comprising both the desired protein and a second protein or other contaminant or impurity mixture, such that at least the majority of the molecules of the desired protein are removed from that portion of the mixture that comprises at least the majority of the molecules of the second protein or other contaminant or mixture of impurities.

[0102] The term “purify” or “purifying” a desired protein from a composition or solution comprising the desired protein and one or more contaminants means increasing the degree of purity of the desired protein in the composition or solution by removing (completely or partially) at least one contaminant from the composition or solution.

[0103] The terms “chromatography media” or “chromatographic medium” refer to any type of stationary phase substrate (solid support), scaffold, or matrix used for chromatography or purification, in which a N-Intein Ligand is affixed, immobilized, bonded, or grafted (covalently or otherwise), for the purpose of separating, enriching, or purifying a secondary molecule of interest. Common examples of chromatography media include but are not limited to: chromatography resins (e.g. crosslinked agarose, polymer, or silica-based particles / porous beads); functionalized membranes; micro- and nano-scale magnetic particles; and structured pore / structured channel media (e.g. monoliths and monolithic columns).

[0104] Disclosures herein relating to immobilization of a N-Intein Ligand upon a “chromatographic medium” are presumed to apply generally to any type of “chromatography media”. The fundamental functional requirement of the “chromatographic medium” is to provide a solid support surface to retain a N-Intein Ligand. As such, it is understood that various chromatographic media may be freely and independently substituted for one another with little or no consequence upon the function of the immobilized N-Intein Ligand.

[0105] Tag-Free Blinatumomab and Related Compositions

[0106] Blinatumomab is commercialized as Blincyto®. A striking and unique characteristic of the approved Blincyto® drug is that it includes a polyhistidine affinity tag (His tag) at its C- terminus (Global Substance Registration Number 4FR53SIF3A, SEQ ID NO: 2). No other drug has ever been approved with an intact affinity tag of any kind, including a His tag. Blinatumomab is a bi-specific T-cell engager antibody with implications in the treatment of relapsed / refractory (R / R) B-cell acute lymphoblastic leukemia (ALL).

[0107] Disclosed herein is a next-generation blinatumomab with significant improvements over the existing molecule. First, this next generation molecule is tag-free (SEQ ID NO: 1), whereas the original molecule was approved by the FDA under the accelerated approval pathway containing a polyhistidine tag at the protein’s C-terminus (SEQ ID NO: 2). Specifically, disclosed herein is tagless blinatumomab, as well as a method of making it, which can utilize the NpuC selfcleaving split intein tag technology for the purification of tagless therapeutic proteins (iCapTag™ platform, Prabhala et al. 2023; Prabhala et al. 2024). This invention therefore overcomes the immunogenicity concerns associated with polyhistidine-tagged proteins. The His Tag provides no therapeutic function or advantage, and in fact may cause complications, as tag-related immunogenicity can cause issues in monitoring patients and adjusting continuous / maintenance dosages.

[0108] Specifically, disclosed herein is a bispecific two single chain antibody comprising an amino acid sequence, wherein said amino acid sequence comprises SEQ ID NO: 1, wherein said amino acid sequence can comprise 1, 2, 3, 4, or 5 variations within its sequence, and wherein the sequence does not comprise any additional amino acids at either its N-terminus or its C-terminus. SEQ ID NO: 1 encodes blinatumomab without a tag.

[0109] Also disclosed herein is a bispecific two single chain antibody comprising an amino acid sequence, wherein said amino acid sequence comprises SEQ ID NO: 1, wherein said amino acid sequence can comprise 1, 2, 3, 4, or 5 variations within its sequence, and wherein the sequence does not comprise a tag at either its N-terminus or its C-terminus, but can comprise other useful components which are discussed below.

[0110] The variations of 1, 2, 3, 4, or 5 amino acids can be consecutive or non-consecutive. They can enhance or increase the effectiveness of blinatumomab in treating disease, or can enhance or increase other desired properties of the molecule. This enhancement or increase in effectiveness can mean an enhancement of it’s therapeutic properties, a reduction in immunogenicity, or an improved safety profile. Further sequence optimization may be employed to increase expression, stability, solubility, or protease resistance, facilitating large-scale manufacturing and / or improving shelf life. In some embodiments, site-specific mutations or linker modifications may also be introduced to optimize the spatial orientation, avidity, or flexibility of the bispecific binding arms.

[0111] As used herein, a “His tag” or “polyhistidine tag” refers to a short stretch of consecutive histidine residues, typically comprising six (Hise) but optionally ranging from 2 to 10 or more histidines, that is genetically engineered into a recombinant protein to facilitate its detection, purification, or immobilization. The His tag is most commonly located at the N-terminus or C- terminus of the expressed protein, but it may also be inserted into internal loops or linker regions, provided that such placement does not disrupt the native folding or function of the protein. The histidine residues exhibit a strong affinity for metal ions such as nickel (Ni2+) or cobalt (Co2+), enabling purification through immobilized metal affinity chromatography (IMAC). In some embodiments, the His tag may be cleavable, such that it can be enzymatically removed following purification.

[0112] While SEQ ID NO: 1 (or a variant thereof) does not comprise a tag on either end, it can comprise other additional components. These can include, but are not limited to, a half life extender, such as an albumin affibody binding protein. SEQ ID NO: 1 (or a variant thereof) can also comprise a signal sequence and a decoy sequence at the N-terminus. These are described in more detail below.

[0113] Half-Life Extenders

[0114] The next generation tagless blinatumomab molecule disclosed herein can comprise a halflife extension fusion protein which significantly improves the serum half-life of the therapeutic protein. Examples of half-life extenders (HLE) include an albumin affibody (ABD). Examples of sequences with an ABD included can be found in SEQ ID NOS: 3, 5, 7, and 8. SEQ ID NOS: 6 and 9 represent ABDs without the associated blinatumomab. Half-life extenders can also be used with the tagged version of blinatumomab (SEQ ID NO: 2).

[0115] Examples of half-life extenders (HLE) include, but are not limited to, Fc-fusion, albuminfusion, and albumin affinity fusion (affibody, nanobody, lipidation, DARPins. Additional methods include PEGylation, PASylation, or glycosylation.

[0116] Specifically, blinatumomab is 54 kDa which is below the renal clearance threshold in vivo. This causes blinatumomab to have a very short half-life of only 2.1 hours in vivo, requiring a continuous infusion of the drug over the course of several weeks for patients undergoing treatment. Albumin affibody fusion protein in the current application helps increase the half-life extension of blinatumomab. This increase can be by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or more compared to a similar molecule without the half-life extender. Put another way, the halflife can be extended by 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, or 1.5 hours, 2 hours, 3, hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, 24 hours, or more.

[0117] Signal Sequences / Decoy Sequences

[0118] Also disclosed herein is tagless blinatumomab (as represented by SEQ ID NO: 1) or a tagged blinatumomab, as represented by SEQ ID NO: 2, with an associated signal sequence / decoy sequence provided. Most mammalian-expressed proteins contain a secretion signal upstream of the gene of interest. This short peptide (-10-30 amino acids) is expressed in front of the protein and then directs it to the endoplasmic reticulum (ER) for processing before being cleaved off by a signal peptidase in the Golgi apparatus, and then finally secreted out of the cell. The rate of endoplasmic reticulum translocation has been identified as a production bottleneck in mammalian expression systems for recombinant therapeutic production (O’Neill 2023).

[0119] There is evidence showing that ER translocation is improved when several residues from a known secretion target are included immediately downstream of the signal peptide. These are referred to as “decoy” sequences. For example, the first 38 amino acids of serum albumin (SA) can be found in SEQ ID NO: 10, where the first 18 residues represent the signal peptide (SEQ ID NO: 11), and the last 20 amino acids represent the secreted 67 kDa SA protein, which serves as the decoy sequence (SEQ ID NO: 12). For recombinant expression, ER translocation is likely improved by including the decoy SEQ ID NO: 12, directly downstream of the signal peptide.

[0120] This decoy strategy is not exclusive to signal peptide / mature protein partners found in nature (such as with the SA example). Work has been done to create highly engineered secretion signals in front of non-natural protein partners. Several examples of such peptides are highlighted by O’Neill et al. These pairings can also be used with the decoy strategy, in which the signal peptide is the engineered molecule, and the decoy is whatever partner protein has been disclosed. For example, “secRecon” is a highly engineered signal peptide developed to improve the secretion of several mammalian- expressed recombinant proteins, including secreted alkaline phosphatase (SEAP). An example of this decoy strategy with this pairing is represented by SEQ ID NO: 13, where the first 21 -residues represent the engineered signal peptide (SEQ ID NO: 14), and the N- terminal 20 amino acids of the 54 kDa SEAP protein follows (SEQ ID NO: 15). The decoy in this example would be SEQ ID NO: 15. Also disclosed is an IgG-kappa consensus signal peptide, SEQ ID NO: 16, with the +5 +10 and +20 of Adalimumab’s heavy chain, SEQ ID NO: 17. Table 1, in the Examples section, shows details for decoys which can be used.

[0121] While these decoy sequences are likely to enhance the recombinant protein secretion, they remain at the N-terminus of the target protein, which is a highly undesirable attribute when producing any therapeutic protein. When traditional protein purification methods are used, it may be possible to remove these extra residues with a protease, however, that adds additional downstream processing steps and expenses. This is not widely performed at manufacturing scales.

[0122] What is significant about this work is that, when iCapTag™ platform is used, the intein tag removes itself and anything upstream of the tag during the purification step, including any decoy sequences left on the secreted protein. This is a significant improvement over the existing technology.

[0123] Nucleic Acids, Cells, and Vectors

[0124] Also disclosed herein are nucleic acids encoding the polypeptides described herein. Specifically, disclosed herein is a nucleic acid encoding any of SEQ ID NOS: 1, 3, 7, or 8. The nucleic acids encoding these polypeptides can be 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOS: 27 and 28, which represent nucleic acids encoding the bispecific two single chain antibodies disclosed herein.

[0125] Put another way, the nucleic acid sequence encoding the amino acid sequence of SEQ ID

[0126] NO: 1 can comprise 10 or more variations within its sequence. In addition, the nucleic acid can encode the additional elements described above, such as the half-life extender and / or the signal sequence / decoy sequence.

[0127] In certain embodiments are antigen-binding constructs produced as recombinant molecules by secretion from yeast, a microorganism such as a bacterium, or a human or animal cell line. In embodiments, the polypeptides are secreted from the host cells.

[0128] Embodiments include a cell, such as a yeast cell transformed to express tagless blinatumomab as described herein. In addition to the transformed host cells themselves, are provided culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium. If the polypeptide is secreted, the medium will contain the polypeptide, with the cells, or without the cells if they have been filtered or centrifuged away. Many expression systems are known and may be used, including bacteria (for example E. coli and Bacillus sublilis . yeasts (for example Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pasloris. filamentous fungi (for example Aspergillus), plant cells, animal cells and insect cells.

[0129] The polypeptide of SEQ ID NO: 1, as described herein, can be produced in conventional ways, for example from a coding sequence inserted in the host chromosome or on a free plasmid. The yeast can be transformed with a coding sequence for the desired protein in any of the usual ways, for example electroporation. Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol. 194, 182.

[0130] Also provided are vectors containing polynucleotides encoding an antigen-binding construct described herein, host cells, and the production of the antigen-binding construct proteins by synthetic and recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.

[0131] Also provided are host cells containing vector constructs described herein, and additionally host cells containing nucleotide sequences that are operably associated with one or more heterologous control regions (e.g., promoter and / or enhancer) using techniques known of in the art. The host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g., a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. A host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically engineered polypeptide may be controlled. Furthermore, different host cells have characteristics and specific mechanisms for the translational and post-translational processing and modification (e.g., phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed.

[0132] Pharmaceutical Compositions and Administration

[0133] Also disclosed are pharmaceutical composition comprising the bispecific two single chain antibody of tagless blinatumomab. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0134] In certain embodiments, the composition comprising the antigen-binding construct is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0135] In certain embodiments, the compositions described herein are formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0136] The amount of the composition described herein which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and / or activity of a therapeutic protein can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses are extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0137] In certain embodiments, an antigen binding construct described herein is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 pg / kg to 15 mg / kg (e.g. 0.1 mg / kg - 10 mg / kg) of T cell activating bispecific antigen binding molecule can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 pg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the antigen binding construct described herein would be in the range from about 0.005 mg / kg to about 10 mg / kg. In other non-limiting examples, a dose may also comprise from about 1 microgram / kg body weight, about 5 microgram / kg body weight, about 10 microgram / kg body weight, about 50 microgram / kg body weight, about 100 microgram / kg body weight, about 200 microgram / kg body weight, about 350 microgram / kg body weight, about 500 microgram / kg body weight, about 1 milligram / kg body weight, about 5 milligram / kg body weight, about 10 milligram / kg body weight, about 50 milligram / kg body weight, about 100 milligram / kg body weight, about 200 milligram / kg body weight, about 350 milligram / kg body weight, about 500 milligram / kg body weight, to about 1000 mg / kg body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg / kg body weight to about 100 mg / kg body weight, about 5 microgram kg body weight to about 500 milligram kg body weight, etc., can be administered, based on the numbers described above. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the T cell activating bispecific antigen binding molecule). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0138] The antigen-binding constructs described herein are generally used in an amount effective to achieve the intended purpose. For use to treat or prevent a disease condition, an antigen-binding construct described herein, or pharmaceutical compositions thereof, are administered or applied in a therapeutically effective amount. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0139] For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. A dose can then be formulated in animal models to achieve a circulating concentration range that includes the IC5o as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.

[0140] Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data.

[0141] Dosage amount and interval may be adjusted individually to provide plasma levels of the antigen-binding construct described herein which are sufficient to maintain therapeutic effect. Usual patient dosages for administration by injection range from about 0.1 to 50 mg / kg / day, typically from about 0.5 to 1 mg / kg / day. Therapeutically effective plasma levels may be achieved by administering multiple doses each day. Levels in plasma may be measured, for example, by HPLC.

[0142] In cases of local administration or selective uptake, the effective local concentration of the antigen-binding construct described herein may not be related to plasma concentration. One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation. Methods

[0143] It is important to note that the production of “tagless” blinatumomab, as described herein, can be used with the self-removing intein process. Therefore, in addition to the tagless blinatumomab itself, also disclosed is a method for making tagless blinatumomab at scale, and various compositions comprising the tagless blinatumomab itself.

[0144] More specifically, the isoelectric point (pl) of blinatumomab is 6.6. Traditional chromatography methods for molecules that do not contain a Fc domain (i.e. traditional monoclonal antibodies (mAb) or mAb-like bi-specific antibodies) often include methods such as ion exchange (IEX) chromatography for separation of the target protein from the host cell proteins (HCPs) and other impurities. Eukaryotic host cells are known to produce HCPs with an average pl point around 7. The fundamental basis of IEX chromatography requires the target molecule to have a significantly different pl from its expression host’s HCP impurities for efficient separation. The proximity of blinatumomab ’s pl to its average eukaryotic HCP pl renders IEX a highly inefficient means of separation in downstream processing. When such a situation arises, the need for specific purification modes such as affinity chromatography are often required. The iCapTag® intein affinity purification of a protein of interest is therefore used for the manufacturing of tagless blinatumomab. The iCapTag® platform can produce tagless blinatumomab, which offers an end product with significant improvement over the current immunogenicity concerns associated with polyhistidine-tagged blinatumomab.

[0145] Therefore, provided herein are methods of producing an expression product containing an antigen-binding construct as described herein, in stable mammalian cells, the method comprising: transfecting at least one mammalian cell with: at least a first DNA sequence encoding said first polypeptide construct and at least a second DNA sequence encoding said second polypeptide construct, such that said at least one first DNA sequence, said at least one second DNA sequence are transfected in said at least one mammalian cell in a pre-determined ratio to generate stable mammalian cells; culturing said stable mammalian cells to produce said expression product comprising said antigen-binding construct.

[0146] Based on this general methodology, described herein is a method of making a bispecific two single chain antibody, the method comprising the steps of: a) providing an amino acid sequence comprising SEQ ID NO: 1, wherein said amino acid sequence can comprise 1, 2, 3, 4, or 5 variations within its sequence, and further wherein the amino acid sequence comprises a Cognate Binding Partner at one of its termini; b) exposing the amino acid sequence of step a) to an N-intein Ligand under conditions that the N-intein ligand and the Cognate Binding Partner form an intein complex; and c) purifying the intein complex of step b); and d) exposing the intein complex to conditions which allow for the Cognate Binding Partner to be cleaved from the amino acid sequence comprising SEQ ID NO: 1 (wherein said amino acid sequence can comprise 1, 2, 3, 4, or 5 variations within its sequence); thereby making a bispecific two single chain antibody. In one embodiment, the amino acid sequence and Cognate Binding Partner can be produced by an expression system prior to step a). Furthermore, the expression system can comrpise a cell transformed with a vector, wherein said vector encodes the amino acid sequence and Cognate Binding Partner. Also disclosed is that the bispecific two single chain antibody can comprise a clarified cell culture supernatant or clarified cell lysate.

[0147] Also disclosed are compositions produced by these methods. For example, the composition can be SEQ ID NO: 1 or a variant thereof as described above. This variant can be substantially pure, such as 90%, 95%, or 100% pure, or any amount below or between these values. An affinity tag may be present in less than 1%, 2%, 3%, 4%, or 5% of recovered antibody molecules.

[0148] Also disclosed is a method of targeting CD3 site on T cells and CD 19 site on B cells, the method comprising exposing the cells to the tagless bispecific two single chain antibody system described herein. This method can be used to treat B cell related conditions, such as lymphoma, B cell leukemia or Hodgkins lymphoma. A specific example includes acute lymphoblastic leukemia (ALL), or B cell precursor ALL. The subject can be either an adult or a pediatric patient (child).

[0149] EXAMPLES

[0150] Several aspects of the expression of intein-tagged blinatumomab have proven challenging, in both upstream and downstream processing. Two cell lines were initially screened, Expi293 and ExpiCHO. ExpiCHO produced a strong Western blot signal at the correct size of NpuC- blinatumomab-his, but also consistently produced strong signals around -35-45 kDa, indicating degradation that was not further investigated at the time. Inconsistent secretion of the protein following the serum albumin (SA) leader sequence in the absence of a decoy sequence was also observed, which led to studies on the removal of SA leader (resulting in cytoplasmic expression) and subsequent lysis of the cell pellet to release the protein into solution. Results from these studies were not strong and the secretion pathway was returned to for production of blinatumomab. The first major success with intein cleavage was observed after manipulating the elution temperature and flanking amino acid identities. Tests on the native blinatumomab sequence with the existing iCapTag™ resin (tag included) showed protein in the expression supernatant but failed to bind strongly or cleave from the resin. A small scar of +MF on the N-terminus of blinatumomab was cloned in and it improved the purification significantly. Blinatumomab can be purified with the native +DIQ starting amino acid sequence. The next generation intein can enable this, and a functional blinatumomab of SEQ ID NO: 1 can therefore be produced.

[0151] DNA sequences for the production of blinatumomab were obtained via gene fragment and cloned into a pTT vector containing a CMV promoter using standard recombinant DNA techniques. Two protein domains, the blinatumomab two single chain variable fragment (scFv) moiety, and the albumin binding domain, were cloned in various orientations to one another and with a few different glycine-serine repeat linkers between domains. Sequences were confirmed by Sanger sequencing reaction from CMV promoter and then secondarily confirmed via full plasmid sequencing (Azenta / Genewiz, NJ). Confirmed plasmids were transformed into E. coli DH5-a (NEB) and maxipreped (QIAGEN) to produce transfection-grade DNA. The DNA was then sterile filtered and used to transfect either Expi293™ or ExpiCHO™ cells depending on the experiment using Gibco’s Expifectamine™ kits (cat. #A14524 for 293, cat. #A29129 for CHO, Thermo Fisher Scientific). The cells were allowed to express the blinatumomab constructs for 4-10 days depending on the experiment before harvest at 4°C, 3,500 xg for 20 minutes in a swinging-bucket rotor.

[0152] After harvest, the supernatant and / or cell pellet was either frozen or held on ice for immediate purification. Note that whether the supernatant or pellet was used is determined by which construct had been expressed. If the supernatant was purified directly, it was gently brought to room temperature and then titrated up to pH 8.5 using 200 mM NaOH. If cell pellet was further purified, the pellet was resuspended in 40% of the initial cell culture volume, sonicated at 4-6W for 3-5 cycles or 10 seconds on 30 seconds off on ice, and clarified at 4°C, 20,000 xg, 5 min. Clarified cell lysate was the diluted into an equal volume amount of pH 8.5 column buffer, gently mixed, and then titrated up to 8.5 using 200 mM NaOH at room temperature.

[0153] A detailed protocol of the intein purification can be found in several published papers (Prabhala et al. 2023; Prabhala et al. 2024). The following description is for a manual / gravity purification performed on bulk resin beads added to an empty gravity column, such as BioRad’s Poly-Prep® Chromatography Column (cat. #7311550). Briefly, an appropriate resin volume was determined based on cell culture volume (generally, -0.25 mL column volume (CV) for a 10 mL expression) and the resin was stripped in either guanidinium-HCL or 150 mM phosphoric acid depending on resin age and previous uses. The column was then equilibrated in pH 8.5 20 mM AMPD 20 mM PIPES 200 mM NaCl column buffer before the protein-containing supernatant or lysis was applied to the column. During binding, the column was capped and allowed to mix on a rotary wheel for 30-60 minutes at room temperature or 60-180 minutes at 4°C. After binding, the resin is washed in pH 8.5 20 mM AMPD 20 mM PIPES 500 mM NaCl buffer, and then the pH is shifted to 6.2 with an ample wash of elution buffer (pH 6.2 20 mM AMPD 20 mM PIPES 200 mM NaCl). The intein cleavage reaction is initiated by the pH shift. The reaction was allowed to proceed for at least 5 hours but no more than 24 hours with regular sampling throughout to track the progression of tagless blinatumomab cleavage from the NpuN-NpuC complex fixed to the resin. After incubation, the tagless blinatumomab was eluted off the column using additional CVs of elution buffer. An optional clean-in-place (CIP) / acid strip step could be performed after all the elutions were collected by adding 1-2 CVs of acid to the capped column and letting it incubate for 30-60 min before collecting and sampling the flow through.

[0154] Several variations of the intein cleavage test can be performed. Some of the reported data in this patent include a Western blot from a study in which a two-residue methionine phenylalanine scar was cloned directly downstream of the intein, before the DIQ natural N-terminus of blinatumomab. This test was performed alongside the non-mutated control, and at both room temperature and 37°C. Additional variations on intein cleavage tests may include changing buffer compositions or changing the elution buffer pH, just to name two possibilities.

[0155] Basic analysis methods for the purification of tagless blinatumomab include SDS-PAGE silver staining and Western blotting. Additional methods to characterize the product include mass spectrometry. Activity assays can be performed in vitro and in vivo. Half-life extension validation can be performed in vivo in mice.

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[0203]

[0047] Bohlen, Cancer Res 57 (1997), 1704-9: Demanet, IntJ Cancer Suppl7 (1992), 67-8 SEQUENCES

[0204] SEQ ID NO: 1: Blinatumomab with no HIS tag

[0205] DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVS GIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGG GSGGGGSQ VQLQQSGAELVRPGS S VKISCKASGYAF S S YWMNWVKQRPGQGLEWIGQ IWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYY YAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMH WVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAV YYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSAS PGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSL TIS SMEAED AAT YYCQQWS SNPLTFGAGTKLELK

[0206] SEQ ID NO: 2: Blinatumomab with HIS tag

[0207] DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVS GIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGG GSGGGGSQ VQLQQSGAELVRPGS S VKISCKASGYAF S S YWMNWVKQRPGQGLEWIGQ IWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYY YAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMH WVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAV YYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSAS PGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSL TIS SMEAED AAT YYCQQWS SNPLTFGAGTKLELKHHHHHH

[0208] SEQ ID NO: 3: Construct: NpuC (removed)-ABD-Blinatumonab (Linker GSG)

[0209] LAEAKVLALRELDKYGVSDYYKDLIDKAKTVEGVKALIDEILAAGSGDIQLTQSPASLA VSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSG TDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQL QQSGAELVRPGS S VKISCKASGYAF S SYWMNWVKQRPGQGLEWIGQIWPGDGDTNYN GKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGT TVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLE WIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHY CLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRA SSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAAT YYCQQWS SNPLTFGAGTKLELK

[0210] SEQ ID NO: 4: NpuC

[0211] H<IATRI<YLGI<QNVYGIGVERDHNFALI<NGFIAHN SEQ ID NO: 5: Construct: NpuC (before removal)-ABD-blinatumomab (Linker GSG)

[0212] IKIATRKYLGKQNVYGIGVERDHNFALKNGFIAHNLAEAKVLALRELDKYGVSDYYKD

[0213] LIDKAKTVEGVKALIDEILAAGSGDIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSY

[0214] LNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQST

[0215] EDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYA F S SYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADES S STAYMQLS S LASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELAR PGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATL

[0216] TTDKS S STAYMQLS SLTSEDS AVYYC ARYYDDHYCLDYWGQGTTLT VS S VEGGSGGS GGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIY DTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

[0217] SEQ ID NO: 6: Albumin affibody binding domain

[0218] FALI<NGFIAHNMFLAEAI<VLALRELDI<YGVSDYYI<DLIDI<AI<TVEGVI<ALIDEILAA

[0219] SEQ ID NO: 7: Construct: NpuC (removed)-blinatumomab-ABD (Linker GSG)

[0220] DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVS GIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGG GSGGGGSQ VQLQQSGAELVRPGS S VKISCKASGYAF S S YWMNWVKQRPGQGLEWIGQ IWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYY YAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMH WVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAV YYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSAS PGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSL TIS SMEAED AAT YYCQQWS SNPLTFGAGTKLELKGSGLAEAK VLALRELDKYGVSD YY I<DLIDI<AI<TVEGVI<ALIDEILAA

[0221] SEQ ID NO: 8: Construct: NpuC (before removal)-blinatumomab- ABD (Linker GSG)

[0222] IKIATRKYLGKQNVYGIGVERDHNFALKNGFIAHNDIQLTQSPASLAVSLGQRATISCKA SQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKV DAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGS

[0223] SVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTAD ESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDI KLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTN YNQKFKDKATLTTDKS S STAYMQLS SLTSEDS AVYYC ARYYDDHYCLDYWGQGTTLT VSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQ

[0224] KSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLT FGAGTKLELKGSGLAEAKVLALRELDKYGVSDYYKDLIDKAKTVEGVKALIDEILAA SEQ ID NO: 9: half-life extending (HLE) albumin affibody (Linker GSG)

[0225] LAEAI<VLALRELDI<YGVSDYYI<DLIDI<AI<TVEGVI<ALIDEILAAGSG

[0226] SEQ ID NO: 10: Signal Sequence Plus Decoy Serum Albumin

[0227] MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDL

[0228] SEQ ID NO: 11: Signal Sequence Serum Albumin

[0229] MKWVTFISLLFLFSSAYS

[0230] SEQ ID NO: 12: Decoy Sequence Serum Albumin

[0231] RGVFRRDAHKSEVAHRFKDL

[0232] SEQ ID NO: 13: secReon Signal Sequence + Secreted Alkaline Phosphatase (SEAP) Decoy Sequence

[0233] MWWRLWWLLLLLLLLWPMVWATACSDSNASNPSDNSASGNP

[0234] SEQ ID NO: 14: secReon Signal Peptide

[0235] MWWRLWWLLLLLLLLWPMVWA

[0236] SEQ ID NO: 15: SEAP Decoy Sequence

[0237] TACSDSNASNPSDNSASGNP

[0238] SEQ ID NO: 16: IgG Kappa Signal Sequence

[0239] MELGLSWIFLLAILKGVQC

[0240] SEQ ID NO: 17: Humeria Heavy Chain Decoy Sequence

[0241] EVQLVESGGGLVQPGRSLRL

[0242] IgG Kappa Signal Sequence + Humeria Heavy Chain Decoy Sequence

[0243] (SEQ ID NO: 16 + SEQ ID NO: 17) TABLE 1: SEQUENCES OF DECOYS

[0244] SEQ ID NO: 27 tagless blincyto alone

[0245] GACATACAGTTGACCCAAAGTCCTGCAAGCCTGGCGGTGAGCCTCGGCCAAAGGGC

[0246] CACCATTTCTTGCAAAGCAAGTCAGTCTGTGGATTATGACGGCGATAGCTATCTCAA

[0247] CTGGTACCAGCAGATCCCCGGACAACCTCCTAAGCTGCTCATTTATGACGCTTCCAA

[0248] TTTGGTGTCCGGAATTCCTCCGCGGTTTTCAGGCAGCGGTTCAGGAACTGATTTTAC

[0249] TCTCAATATCCATCCTGTTGAGAAGGTCGACGCAGCAACTTATCATTGTCAGCAGTC

[0250] AACTGAAGACCCATGGACCTTCGGCGGCGGAACCAAACTTGAGATAAAAGGTGGA

[0251] GGAGGTTCAGGCGGAGGAGGAAGCGGCGGGGGAGGAAGCCAGGTGCAGCTCCAAC

[0252] AGTCCGGGGCAGAGTTGGTCCGCCCTGGATCAAGCGTTAAAATTTCCTGTAAAGCG

[0253] TCTGGATATGCTTTCAGTTCTTATTGGATGAATTGGGTCAAGCAGCGACCAGGGCAA

[0254] GGGCTGGAATGGATAGGCCAGATCTGGCCAGGGGATGGCGATACAAATTACAACG

[0255] GCAAGTTTAAAGGTAAAGCTACATTGACAGCCGATGAAAGCTCTTCCACCGCATAT

[0256] ATGCAGCTCAGCTCACTCGCCAGCGAGGATAGTGCGGTCTATTTTTGTGCTCGCCGC

[0257] GAGACCACAACTGTCGGCAGATATTATTATGCTATGGATTATTGGGGGCAGGGGAC

[0258] AACCGTCACTGTTAGTTCTGGGGGAGGGGGGAGCGACATCAAACTCCAGCAATCTG

[0259] GGGCGGAATTGGCGCGGCCTGGCGCCTCCGTGAAGATGTCATGTAAGACTTCTGGT

[0260] TATACTTTTACTCGCTACACGATGCACTGGGTCAAGCAGAGACCCGGGCAAGGACT

[0261] CGAGTGGATCGGGTACATTAACCCCTCTCGGGGGTATACGAATTACAACCAAAAAT

[0262] TCAAGGACAAGGCCACCCTTACGACCGATAAGAGCAGCAGCACGGCCTACATGCAA

[0263] CTTTCTTCCTTGACCTCTGAGGATTCCGCTGTTTACTATTGCGCGCGGTACTACGACG

[0264] ACCATTATTGTCTCGATTATTGGGGACAGGGCACTACTTTGACTGTGAGTAGCGTTG

[0265] AGGGGGGTTCTGGGGGATCTGGGGGAAGTGGCGGGTCAGGCGGGGTAGATGACAT ACAACTGACCCAGAGTCCCGCCATAATGTCCGCTTCCCCGGGAGAAAAAGTTACCA

[0266] TGACCTGCCGCGCGTCTTCCAGCGTATCTTATATGAATTGGTATCAGCAGAAAAGCG

[0267] GAACCTCCCCTAAACGCTGGATATATGACACCAGCAAGGTGGCATCTGGCGTACCC

[0268] TACCGCTTTTCAGGATCAGGCTCAGGAACTTCCTATTCTCTTACGATCAGTAGTATG GAGGCCGAAGATGCTGCGACTTACTACTGCCAGCAGTGGTCCAGTAACCCTCTCAC

[0269] ATTTGGGGCAGGTACGAAGCTCGAATTGAA

[0270] SEQ ID NO: 28: albumin affibody DNA sequence

[0271] CTCGCCGAGGCTAAAGTTCTGGCCCTCAGAGAACTGGATAAATACGGAGTATCAGA TTACTACAAAGACCTTATAGACAAGGCAAAGACGGTAGAAGGAGTCAAGGCGTTG

[0272] ATCGACGAAATCCTCGCCGCC

Claims

CLAIMSWhat is claimed is:

1. A bispecific two single chain antibody comprising an amino acid sequence, wherein said amino acid sequence comprises SEQ ID NO: 1, wherein said amino acid sequence can comprise 1, 2, 3, 4, or 5 variations within its sequence, and wherein the sequence does not comprise any additional amino acids at either its N-terminus or its C-terminus.

2. A bispecific two single chain antibody comprising an amino acid sequence, wherein said amino acid sequence comprises SEQ ID NO: 1, wherein said amino acid sequence can comprise 1, 2, 3, 4, or 5 variations within its sequence, and wherein the sequence does not comprise a tag at either its N-terminus or its C-terminus.

3. The bispecific two single chain antibody of claim 1 or 2, wherein the sequence further does not comprise a His tag or other conventional affinity tag at either its N-terminus or its C-terminus.

4. The bispecific two single chain antibody of claim 2, wherein the antibody further comprises a half-life extender.

5. The bispecific two single chain antibody of claim 4, wherein the half-life extender comprises an albumin affibody binding protein.

6. The bispecific two single chain antibody of claim 2, wherein the antibody further comprises a signal sequence and a decoy sequence at the N-terminus.

7. A pharmaceutical composition comprising the bispecific two single chain antibody of any one of claims 1-6.

8. A nucleic acid encoding the bispecific two single chain antibody of claim 1 or 2.

9. A vector comprising the nucleic acid sequence of claim 8.

10. An amino acid sequence consisting of SEQ ID NO: 1, 3, or 7.

11. An amino acid sequence consisting of SEQ ID NO: 1, 3, or 7, and a half-life extending peptide.

12. The amino acid sequence of claim 11, wherein the half-life extending peptide comprises albumin affibody binding domain.

13. A pharmaceutical composition comprising the amino acid sequence of any one of claims 10-12.

14. A nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1, wherein the nucleic acid can comprise 10 or more variations within its sequence, and wherein the nucleic acid sequence further encodes a C-terminal intein segment.

15. A vector comprising the nucleic acid of claim 14.

16. A method of making a bispecific two single chain antibody, the method comprising the steps of: a. Providing an amino acid sequence comprising SEQ ID NO: 1, wherein said amino acid sequence can comprise 1, 2, 3, 4, or 5 variations within its sequence, and further wherein the amino acid sequence comprises a Cognate Binding Partner at one of its termini; b. Exposing the amino acid sequence of step a) to an N-intein Ligand under conditions that the N-intein ligand and the Cognate Binding Partner form an intein complex; c. Purifying the intein complex of step b); and d. Exposing the intein complex to conditions which allow for the Cognate Binding Partner to be cleaved from the amino acid sequence comprising SEQ ID NO: 1 (wherein said amino acid sequence can comprise 1, 2, 3, 4, or 5 variations within its sequence); thereby making a bispecific two single chain antibody.

17. The method of claim 16, wherein the amino acid sequence and Cognate Binding Partner are produced by an expression system prior to step a).

18. The method of claim 17, wherein the expression system comprises a cell transformed with a vector, wherein said vector encodes the amino acid sequence and Cognate Binding Partner.

19. The method of any of claims 16-18, wherein the Cognate Binding Partner comprises a C- terminal intein segment.

20. The method of claim 16, where the bispecific two single chain antibody of claim 1, 2, 3 or 4 comprises a clarified cell culture supernatant or clarified cell lysate.

21. A composition produced by the method of any one of claims 16-20.

22. The composition of claim 21, wherein SEQ ID NO: 1 is substantially pure.

23. The composition of claim 21, wherein recovered SEQ ID NO: 1 is 95% or more pure and an affinity tag is present on less than 1% of the antibody molecules.

24. The bispecific two single chain antibody of claim 1, 2, 3 or 4, where the bispecific two single chain antibody produced by the method of claim 16 is at least 95% pure and an affinity tag is present on less than 1% of the antibody molecules.

25. A method of targeting CD3 site on T cells and CD 19 site on B cells, the method comprising exposing the cells to the bispecific two single chain antibody of any one of claims 1-6.

26. A method of treating B cell-related conditions in a subject thereof, the method comprising exposing the subject to the bispecific two single chain antibody of any one of claims 1-6.

27. The method of claim 26, wherein the B cell related condition comprises lymphoma, B cell leukemia or Hodgkins lymphoma.

28. The method of claim 27, wherein the subject has acute lymphoblastic leukemia (ALL).

29. The method of claim 28, wherein the subject has B cell precursor ALL.

30. The method of any one of claims 26-29, wherein the subject is an adult or a child (pediatric patient).

31. A kit comprising the bispecific two single chain antibody of any one of claims 1-6.

32. A bispecific two single chain antibody comprising an amino acid sequence, wherein said amino acid sequence comprises SEQ ID NO: 2 and an albumin affibody binding protein.

Images