WO2020015805A1 - A bi-specific antibody to target cd176 and interleukin-1 receptor associated protein on human cancer stem cells. - Google Patents

Abstract

Disclosed herein is a bi-specific antibody that specifically targets tumor antigen of the cancer cell and cytokine receptor and thereby killing cancer cells. The bi-specific antibody of the present disclosure includes a first antigen binding site that binds to Thomsen-Friedenreich antigen (TF, or CD 176) a tumor-associated carbohydrate epitope and a second antigen binding site that binds to a target membranous protein, Interleukin one receptor accessory protein (IL1RAP). The antibody selectively bound to leukemias tumor cells expressing CD 176 and IL1RAP to kill the target cancer cells, in vitro but not corresponding normal cells which in turn may trigger the immune system to induce cell death (e.g. by CDC).

Description

A Bi-specific antibody to target CD176 and interleukin-1 receptor

associated protein on human cancer stem cells.

Technical Field :

The present invention relates generally to antibodies against tumor markers, for therapeutic use, for example; immune therapy , diagnostic use, for example, identifying cancer stem cell burden and research use, for example isolating cancer stem cells for in vitro studies .More particularly, the invention relates to antibodies against Thomsen-Friedenreich antigen (TF, or CD 176), specifically bi-specific antibodies, that recognize a TF peptide and interleukin receptor accessory protein (IL1RAP).

Background Art :

Cancer stem cells are accused for being responsible for cancer drug resistance which

ultimately leads to treatment failure, metastases and recurrences (Prieto-Vila et al., 2017) making therapeutic approaches for targeting CSCs a crucial need (Phi et ah, 2018).

Accordingly, the present invention seeks to provide new agents and methods for use in

the treatment, diagnosis and study of both solid and hematologi caltumours.

Immunotherapy is now the fourth pillar of cancer therapy, with surgery, radiation, and

traditional chemotherapy being the remaining pillars(McCune 2018). Evasion of the host immune system is a key mechanism to promote malignant progression. Therapeutically

targeting immune pathways has radically changed the treatment paradigm for solid and

lymphoid tumors but has yet to be approved for myeloid malignancies(Przespolewski et ah, 2018). Using monoclonal antibodies(mAb) as a tool for cancer therapy, still has its

limitations. Patients who receive mAh therapy may develop drug resistance or fail to

respond to treatment owing to the multiple signaling pathways involved in the

pathogenesis of cancer and other diseases(Fan et ah, 20l5).Targeting more than one

molecule has proven to circumvent the regulation of parallel pathways and avoid

resistance to the treatment (Varela 2015). A characteristic cell surface marker have been investigated; Thomsen-Friedenreich antigen (TF, or CD 176) a tumor- associated

carbohydrate epitope. The CD 176 antigen was found to be expressed on the surface of

various cancer initiating cells, such as breast, colorectal carcinomas, hepatocellular carcinomas (HCC) , several leukaemias, and other types of cancer, but absent from almost all normal adult cell types. CD176 was also found to be expressed on the surface of CD34+hematopeitic stem cells of the K562 erythroblastic leukemia cell line ; a cell line derived from CML patients. Being strongly expressed on the surface of cancer cells and virtually absent from normal tissues, CD 176 was assumed to be a suitable target for cancer biotherapy with the development of anti-CD 176 antibody that induced apoptosis of leukemic cells (Goletz et al., 2003). Another characteristic marker is the called IL-1 receptor accessory protein (IL-lRAcP), a Toll-like receptor super- family member and a co-receptor of interleukin 1 (IL1R1) (Jaras et al., 2010), that contains three Ig-like C2- type domains in the extracellular region and a long cytoplasmic domain implicated in signal transduction. Two alternative splice variants of IL-1RAP have been identified: membrane- bound IL-1RAP (mIL-lRAP, iso form 1) promotes intracellular IL-1 signaling whereas soluble IL-1RAP (sIL-lRAP , isoform 2 ) contributes to the antagonism of IL-l action by the type II decoy receptor contributing to its regulation. As an indispensible molecule in the IL-l receptor

signal transduction, IL-1 RAP is necessary to link events on the plasma membrane level to downstream signaling pathways (Ali et al.,2007). Blockade of tumor inflammation has potential for cancer therapy, not only provides a primary mechanism to inhibit tumor growth but also as adjuvant to other therapeutics (Liberg et al., 20l8).Using antibodies against IL1RAP was found to be capable of blocking IL-l signaling as well as inhibiting tumor cells growth in AML(Agerstam et al., 2015), breast cancer (Liberg et al., 2018), CML(Jaras et al., 2010) , prostate cancer, breast cancer, lung cancer, colorectal cancer, melanomas, bladder cancer, brain/CNS cancer, cervical cancer, oesophageal cancer, gastric cancer, head/neck cancer, kidney cancer, liver cancer, lymphomas, ovarian cancer, pancreatic cancer, and sarcomas (US9403906B2 ) especially in cancer stem cells, or CSCs) and progenitor cells, which are responsible, directly or indirectly, for the development of a solid tumour in an individual(EP2665749Al ).With referral to the data mentioned above, CD 176 and IL1RAP antigens, were found to be expressed on cancer stem cells, of multiple cancers, each separately. In our disclosure, we sought to identify and target the co- expression of both markers on cancer cells of a hematological tumor, for example the CML and specifically cancer stem cells, as a model for disease persistence. Bi-specific antibodies, are single molecules capable of recognizing 2 targets. The majority of bispecific antibodies are generated by genetic engineering. The dual specificities are conferred by each Fab, requiring a classical IgG assembly but with 4 distinctive polypeptide chains (2 different LCs and 2 different HCs). Random chain associations, namely LC mispairing and unwanted HC homodimerization, initially hindered the development of such bispecific antibodies, but these drawbacks have been progressively overcome by judicious genetic engineering. The application of a common light chain, orthogonal Fab interface (complementary mutations introduced at LC and HC interface in one Fab and no change to the other Fab) or CrossMab (one Fab with switched VH or CH1 domain(s) with the partner VL or CL domain(s) and the other Fab untouched) have essentially eliminated the mispairing of LCs (Kontermann 2012). For HC mispairing, a number of engineering strategies in CH3 domains have also significantly enhanced HC hetero- dimerization based on steric or electrostatic complementarity. Among them, the most successful and widely applied route is knobs-into-holes, in which a“knob” is created by replacing T366 with a bulky residue W on one HC, and the corresponding“hole” is made by triple mutations of T366S, S354C , L368A and Y407V on the partner HC(Merchant et al., 1998).

Disclosure Of The Invention :

The disclosure describes novel bi-specific antibody that targets CD 176 antigen and IL1RAJP. Methods of using the novel antibody can comprise depletion of cancer stem cells from the bone marrow before bone marrow transplantation in leukemic patients, in order to protect those patients from relapses, or as an adjuvant therapy to current anticancer treatment of leukemia, in order to prevent relapses in leukemia patients. Added to this, previously reported expression of either markers in multiple solid tumors; prostate and breast carcinomas and other some types of lymphomas, can provide a universal protection from relapses and metastases in hematological versus solid tumors. And finally, isolation of the cancer stem cells positive for both markers; CD176 and IL1RAP, for further studies of underlying pathways and disease mechanisms can be a research application. Bispecific antibodies(Bis-Abs) are antibodies that can recognize two different epitopes. In one aspect, they can interact with two different cell-surface antigens instead of one with subsequent boosting of the binding specificity (Fan et ah, 2015). FIG.l shows the mechanisms of action of Bis-Abs; they can redirect specific immune cells to the tumor cells to enhance tumor eradication, enable the simultaneous blocking of two different targets that have common signaling pathways, in addition to the above mentioned mechanism.

In one aspect of the present disclosure, by using FACS gene expression analyses of primary CML cells, we identified IL1RAP and CD 176 as candidate targets. This result is supportive to the fact that both antigens are involved in the apoptotic pathways, where IL1RAP up-regulation was associated with decreased apoptosis in AML(Barreyro et ah, 2012) while anti-CDl76 antibody was found to induce apoptosis of CD 176-positive leukemic cells through the involvement of five pathways: The CD95 signaling pathway, the DR3 and DR4/5 death receptor pathway, caspase cascade in apoptosis, the mitochondrial signaling pathway, and apoptotic DNA fragmentation and tissue homeostasis (Yi et ah, 2011).

By using Flow-drop-FISH and CFU assays, IL1RAP was used for the separation of BCRABL+ and BCR-ABL - CML CD34+CD176+ stem cells. Testing the stem-cell characteristics of these two cell populations in immune-deficient mice would have been advantageous, yet, the low numbers of sorted CML cells acquired from the IL1RAP- and IL1RAP+ CD34+CD176+ cell subpopulations, together with the general low engrafting efficiency of chronic phase CML cells in these mice (Jaras et ah, 2010), prevented us from successfully performing such experiments.

FIG.2 showing how CD 176 and IL1RAP antigens are co-expressed on CD34⁺Leukemia stem cells.

In another aspect, in this disclosure, in order to generate a bi-specific antibody that targets CD 176 and IL1RAP, we are unique in providing proof of concept that CML CD34+CD176+ IL1RAP+ cells can be targeted using our bi-specific antibody, while preserving corresponding normal cells. The efficient production of Bis-Ab IgG in single host cells is highly desirable to support the clinical development of these complex molecules. Here, we used the orthogonal Fab design; CHl_Hl72A_F174G and CL_Ll35Y_Sl76W (Lewis et al.,20l4) to facilitate selective Fab arm assembly along with previously described knobs-into-holes mutations (Ridgway et al., 1996)( Atwell et al.,l997) for preferential HC hetero-dimerization. One of the biggest challenges in cloning individual HCs and LCs is the selection of recombinant constructs on antibiotic agar plates and subsequent colony screening. To facilitate the cloning process, these steps were eliminated by applying a highly efficient ligation-independent In-Fusion HD cloning kit. In the In-Fusion HD reaction, reactions enhance homologous recombination between the vector and insert to circularize the vector, thus, little to no background is achieved. In our study, it was found that there was little to no background colonies resulting from undigested plasmid when the vector was linearized with EcoRI restriction enzyme that cut between the leader and C region sequences in addition to the percentage of clones with inserts that varied from 20% to 100% per transformation for HC plasmids and an average 50% for LC plasmids (data not shown) which was relatively consistent with the percentage range of 30%-l00% for HC plasmids versus 81.1% for LC plasmids (Jared et al.,2l06).

A third aspect in the current disclosure, we generated a duel positive cell line; KG1/RAP, in order to validate our bi-specific antibody. In doing so, we used lentiviral transduction. KG1 cell line is a CD176+IL1RAP low/absent expressing cell line. pHRST lentiviral transduction was chosen owing to successful protein expression induction in both immune (Pan et al.,2007) and leukemic cells (Biagi etal.,200l).

A further aspect of the disclosure is that, we have not directly demonstrated that the complement- dependent cytotoxicity (CDC) effect exclusively targets the CD 176+ IL1RAP+CML cells within the CD34+ cell subset. However, given that on average, 50% of the cells within the CD34+ subpopulation in the patients tested were CD176+ IL1RAP+ and the almost undetectable CDC in CD34+ cells from normal controls, our data strongly support the idea that the CD176/IL1RAP targeting antibody (TF/RAP) indeed induce CDC preferentially in CD176+ IL1RAP+ CML CD34+ cells. That potential is supported by studies on multiple cell surface antigens that demonstrated increased or synergistic CDC effect by certain non-cross blocking antibody

combinations and was demonstrated to increase complement activation of CD20 antibodies (Joost et al., 2015). Therapeutic antibodies are commonly administered intravenously, yet selectivity and specificity is a major concern for reducing toxicity. CD176/IL1RAP expression was not present in monocytes unlike the reported weak but present IL1RAP expression in monocytes (Jaras et ah, 2010). Both antigens were low or absent in most types of normal bone-marrow progenitor and mature cell types, suggesting that CD176/IL1RAP dual targeting antibodies are expected to show low toxicity on normal hematopoietic cells. It is noteworthy that although the antibody mode of action in CDC, is to direct immunological cells to target cell killing, the therapeutic mechanism here might be independent of known mechanisms of TKI resistance in CML. Therefore, we hypothesize that the concept of complement-mediated killing of IL1 RAP/CD 176 expressing CML cells may also have the potential to eradicate such cells in patients, either alone or in combination with current regimens, leading to an ultimate cure for CML patients. Finally, our bi- specific Ab (TF/RAP), is not only going to eradicate the CD176+IL1RAP+CML resistant stem cells but also has the potential of being a universal therapeutic for preventing relapses in both types of cancers; solid and hematological owing to the common apoptotic pathways they both share. Further studies need to be elicited in order to confirm the coexpression of both markers, especially in resistant and relapsing cancer patients.

The present disclosure is exemplified with respect to the examples and figures below. However, this is exemplary only, and the disclosure can be broadly applied to target CD176 and IL1RAP simultaneously, on any cancer cells, expressing bothmarkers.

The following experiments and examples are intended to be illustrative only, and not unduly limit the scope of the appended claims.

Example 1 :

In order to evaluate the co-expression of IL1RAP and TF/CD176 antigens on the surface of CD34+ hematopoietic stem cells (HSCs), flow cytometric analysis was done on the PBMCS of CML patients as compared to the PBMCs of the healthy donor. After validating the co- expression of both antigens on the CD34+ cells, progenitor activity was evaluated in flow- sorted CD34+ CD176+IL1RAP+ cell population versus CD34+CD176+IL1RAP- using the Colony Forming Unit (CFU) assay. Subsequent FISH analysis for bcr-abl expression was evaluatedin both

populations. Methods:

PBMCs from CML patients (Oncology Research Gundersen BioBank, WI, USA). For CML patient characteristics, see Table 1. As reference samples, peripheral blood samples from healthy volunteers were obtained after informed consent (Gulf Coast Regional Blood Bank,

Housten, TX, USA). Flow cytometric analyses were performed in a BD LSR Fortessa™), and flow cytometric cell sorting was done in a FACSAriall (P0010) cell sorter. Both are from BD Biosciences, USA. Flow antibodies used were as follows :Primary TF/CD176 anti-CD 176 mAb mouse IgM; (Glycotope, Berlin, Germany ), secondary antibody for TF/CD176 (FITC)- conjugated anti-mouse IgM (m-chain specific, #F9259; Sigma ), primary human CD34 APC- conjugated Monoclonal Mouse IgGl Clone # QBEndlO antibody (FAB7227A-025, R and D systems), Primary Human IL-1 RAcP/IL-l R3 PE-conjugated antibody. K562 cell line(ATCC# CCL- 243)were maintained in RPMI1640 (Life technologies, INC) supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 pg/ml streptomycin sulfate at 37°C in a humidified 5% C02 incubator.

Table 1.CML patients characteristics.

ID Sam A Sex Race BCR Treatment Lifespa p le g e n

Type ABL

C PB 75 Fema Whit + Imatinib j eceased

M M C le e

L-l

CM PBM 70 Male Whit Gleevac Deceased

L-2 C e 2

C PB 53 Fema Whit + Naive Alive ³

M M C le e

L-3

C PB 76 Fema Whit Hydroxyur Alive ³

¹ Date of the latest information:March,l^st 2018.

9 CML-l and CML-2 patient were deceased 5 and 4 years respectively after diagnosis.

^JPatient were alive the time of this

disclosure. Results:

In order to analyze the co-expression of CD 176 and IL1RAP antigens on peripheral blood from a normal volunteer, CML patients, and K562 cells, were isolated and stained with, anti-CDl76, and anti-ILlRAP monoclonal antibodies and analyzed using FACS. CD34+ cells, were also checked (FIG 2). CD34+ cells expression ranged from 7% - 38% in CML samples versus 83.7% in K562 cells (Figure 2A, upper panel). Within the CD34+ cell population, CD 176 and IL1RAP antigens were variably expressed in CML samples, ranging from 1.75% in CML-4 up to 50% in

CML-l, while in K562 cell line, CD 176+ IL1RAP+ cells were detected in 78% of CD34 cells. CML1 to CML4 contained 9% to 50% of CD176+ IL1RAP- cells (FIG 2A, lower panel). CD34+ cells revealed higher frequency of CD 176+ IL1RAP+ in CML group compared to control sample (17.5% versus 3.4%, /?<0.00l)(Fig2B) Surface co-expression of CD 176 and IL1RAP was not only detectable on CML CD34+ cells in BCR-ABL positive patients, but was also demonstrable in the only patient with negative BCR- ABL(CML-2) as well (FIG.2C).

FIG.2 shows peripheral blood mononuclear cells (10³-10⁵ cells) of CML patients (n = 4) and healthy volunteers (n = 1) were isolated and stained for flow cytometry analysis. A. FACS Dot Blot showing the co-expression of CD176 and IL1RAP antigens on the CD34+cells in CML PBMCs compared to NPBMCs. CD34+cell gate was identified then CD 176 and IL1RAP were plotted. B. Statistical analysis of levels of expression CD34, CD34⁺CDl76⁺ILlRAP⁺subsets in normal versus CML patients respectively, /? < 0.01. C. Bar graphs showing the percentage of IL1RAP and CD 176 expression in CD34⁺subsets from CML patients previously assessed for BCR-ABL expression as compared to control. The error bars represent the variation in two independent experiments. D. Bar graphs showing the average count of colony forming units (CFU) that formed two weeks after sorting 1000 CD34⁺ hematopoietic stem cells in CML-2 and CML-4 samples.*/? < 0.05, **/? < 0.01, n.s represents that there is no significant difference between groups.

CD34+CD176+ILlRAP+cell population are Colony-forming cells

In order to analyze the progenitor activity of CML-2 and CML-4, samples were flow sorted for the two populations; CD34+CD176+IL1RAP+ and CD34+CD176+IL1 RAP- then tested for CFUs. The CFU score in CD34+CD176+IL1RAP+ represented 30% of the sorted cells with a significant difference between both populations in CML-4, p<0.0l(FIG.2D). All the sorted populations for both samples were FISH analyzed, only the CD34+CD176+IL1 RAP-showed 40/190 score for BCR-ABL (FIG.3).

Example 2:

Generation of TF/RAP; the bi-specific anti-Human CD176/IL1RAP Antibody.

Methods:

a. Generation of mutated constant domains.

pLM2 plasmids, for both light and heavy chains, were used as templates. PCR conditions used were forty cycles of 94c for 10 seconds, 60 c for 45 seconds and 72c for 5 minutes using Deep Vent Polymerase (New England Bio labs, INC) and 0.5nM primers concentration. Sequences for primers and fusion fragments lengths are listed in Table 2. The mutated

fragments were then gel purified and sub-cloned into their corresponding vectors using restriction enzymes according to standard protocols (Table 3). Colonies were then verified by double restriction digestion and sequencing

b. Synthesis of Variable domains.

For CD176, two clones having the most conserved amino acid sequences were chosen, from the published patent (WO2011089004 Al) Clone 1; VH 1 and VL1, Clone 2; VH2 and VL2. For IL1RAP, Two clones having the most consistent amino acid sequences were chosen, from the published patent (US 20150315279 Al) Clone 4B6; VH and VL and Clone 4G9; VH and VL. Variable domains (VD) were synthesized in order to be compatible with Infusion Cloning. Fifteen base pairs of homologous sequences were added at both the 3’and 5’ ends of both antibodies; CD 176 and IL1RAP and flanked with EcoRI restriction enzyme site. Using the Gene Art online tool, sequences were codon optimized to be expressed in homo sapiens. The sequences were purchased in the form of plasmids (Table 4).

c. Infusion cloning of variable domains into mutated vectors.

An In-Fusion HD cloning kit (Clontech, Inc) was used to clone the V regions of either antigen between the leader and C regions in EcoRI enzyme-digested pLM2-leader-C region vectors. Colonies verified by PCR and restriction mapping showed positive sequencing results.

d. Transient transfection and Bi-specific antibody production in HEK293T cells.

Adherent HEK cells were transfected as above. A total of 14 pg high-quality plasmid- DNA, 10%, GFP plasmid for assessment of transfection efficiency while the rest were heavy and light chain plasmid DNA, combined at a ratio of 1 :1. Six to eight hrs later, cells were gently washed once with PBS and fresh growth medium added. Sixteen hrs post-transfection, the medium was replaced with DMEM supplemented with 5% FCS and incubated at 5% C02 for 24 hrs prior to the initial collection of antibody supernatant. A second collection was made after a further 24 hrs. Third of the conditioned medium from the two harvests were combined and cleared by centrifugation at 1500 rpm for 5 min at 4°C then passed through a 0.45 pm pore PVDF Millex-HV filter (Millipore). FIG.4 illustrates the four bi-specific antibody arms.

A. Site-directed mutagenesis using polymerase chain reaction overlap extension. B. Cartoon showing the bi-specific antibody arms, transfected into HEK cell line;

TFl;CDl76_VHl_CHl_Hl72AFl74G_CH2_CH3T366W ,

CDl76_VLl_CL_Ll35Y_Sl76W, TF2;

CD176_VH2_CHl_Hl 72AF174G_ CH2_CH3T366W,

CD 176_VL2_CL_Ll 35 Y_S 176 W, RAP a; ILlRAP_4B6VH_CHl_CH2_CH3S354C, IL1RAP 4B6VL CL,

RAP b; ILlRAP_4B9YL_CL,ILlRAP_4B9VH_CHl_CH2 CH3S354C.

TF: Thomsen - Freidenrich (CD176 antibody), RAP; IL1RAP antibody, K; Knob, H;

Hole, Y; L135Y mutation, W; S176W mutation, A; H172A, G; F174G, VH; Variable domain heavy chain, VL; Variable domain light chain. See Table 5, for full amino acid sequences of the four arms of the bi-specific antibody and Table 6 for the CRDs of CD176 and IL1RAP antigens.

Table 2: Sequences of primers used for mutagenesis.

*The two mutations in the H172A_F174G were included in the same primer for their proximity Bold text shows the sequences of the overlapping parts of the inward primers.

Table 3: Lengths of generated overlapping and fusion fragments.

Table 4 : Variable domains (VD) sequences for the multiple arms variations.

TF; Thompsen- Freidenrich (CD176),TF1;TF Fab sequence #1, TF2; TF Fab sequence #2, RAP;IL1RAP ,RAP a; Fab sequences of clone 4B6,RAP b; Fab sequences of clone 4G9.Blue and Yellow highlighted; 15bp overlap on the pLM2 IgG plasmid. Underlined: EcoRI sequence; GGATTC

Table 5:Full amino acid sequences for the four arms of bi-specific

antibody: SEQ ID NO:

1.CD 176_VH 1 _CH 1 _H 172AF 174G_CH2_CH3T366W METDTLLLWVLLLWVPGSTGDQVQLKQSGAELVRPGTSVKISCKASGYTFTNY

WLGWVKQRPG

HGLEWIGDIYPGGGYTNYNEKFKGKATLTADTSSSTAYMQLSSLTSEDSAVYFC

AYYDAAGPWF

AYWGQTTLTVSSDISSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS

WNSGALTSGV

ATGPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THT CPPCP APEL

LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK

TKPREEQYNST

YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP

SREEMTKNQVS

LWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVM HEALHNHYTQKSLSLSPGK SEQ ID NO: 2.CDl76_VLl_CL_Ll35Y_Sl76W

METDTLLLWVLLLWVPGSTGDDVLMTQTPLSLPVSLGDQASISC

RSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE DLGV YYCFQGSHVPYTFGGGTKLEIKRADISSTMGTKLTVLGQPKA APSVTLFPPSSEELQANKATLVCYISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYA AWS YLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO: 3. ILlRAP_4B6VH_CHl_CH2_CH3_S354C

METDTLLLWYLLLWVPGSTGDEVKLVESGGGLVKPGGSLKLSCAASGFTFRTYAMSWVRQ

TPEK

RLEWVASISSRGRIYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARDGDYGSSY

DYW

GQGTTLTVSSDISSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG

VHTF

PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

LLGG

PS VFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYN S TYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQV

SLTC

LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEAL HNHYTQKSLSLSPGK SEQ ID NO: 4. ILlRAP_4B6VL_CL

METDTLLLWVLLLWVPGSTGDDIQMTQSPSSLSASLGERVSLTCRASQDIGSSLNWLQQEPD

GTIK

RLIYATSSLDSGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQYASSPWTFGGGTKLEIKDI

SSTM

GTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVET TTPS KQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO: 5. CDl76_VH2_ CHl_Hl72A_Fl74G_T366W

METDTLLLWVLLLWVPGSTGDQVQLKQSGAELVRPGTSVKISCKASGYTFTNYWLGWVK QRPG HGLEWIGDIYPGGGYTNYNEKFKGKATLTADTSSSTAYMQLS SLTSEDSAVYFCAYYDAAGPWFAYWGQTTLTVSSDISSASTKGPSVFPLAPSSKSTSGGTAA LGCL

VKDYFPEPVTVSWNSGALTSGVATGPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS

NTKV

DKKVEPKSCDKTHTCPPCP APELLGGPS VFLFPPKPKDTLMISRTPEVT CVVVDV SHEDPEV KFNW

YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS

KAKG

QPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 6. CD176 VL2_Ll35Y_Sl76W

METDTLLLWVLLLWVPGSTGDDIQMTQSPLSLPVTPGEPASISCRSSQSIVHSNG

NTYLEWYLQLPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGV

YYCFQGSHVP

YTF GGGTKVEIKRADI S STMGTKLTVLGQPKAAPS VTLFPP S SEELQ ANKATLV C Y ISDFYPGAVTV AWKADSSPVKAGVETTTPSKQSNNKYAAWS YLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO: 7. ILlRAP_VH_4G9_CHl_CH2_CH3_S354C

METDTLLLWVLLLWVPGSTGDOVOLOOSGTELVRPGASVKLSCKASGYTFTDYEMHWYK

QTPV

HGLEWIGAIDPGTGGIAYNQKFKGKATLTADKSSSTAYMELRSLTSEDSAVYYCTLYDYDL

AYW

GQGTLVTVSADISSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS

GVHT

FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP

ELLG

GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN

STYR

VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQ

VSLT

CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEA LHNHYTQKSLSLSPGK SEQ ID NO: 8. ILlRAP_VL_4G9_CL

METDTLLLWVLLLWVPGSTGDDVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEW

FLQK

PGQSPKLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPPTFGGGT

KLEI

KDISSTMGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSP VKAG VETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

Table 6: CRD sequences

CD 176 CRD sequences

(Patent WO2011089004

Al)

Heavy chain CDRs

CDR-H1-SEQ ID N0.9: NYWLG

CDR-H2-SEQ ID NO.10:

DIYPGGGYTNYNEKFKG CDR-H3-SEQ

ID NO.11 : YDAAGPWFAY

Light chain CDRs CDR-L1-SEQ ID N0.12:

RSSQSIVHSNGNTYLE CDR-L2-SEQ

ID NO.13: KVSNRFS

CDR-L3-SEQ ID N0.14: FQGSHVPYT

IL1RAP CRD sequences

( Patent US 20150315279 Al)

4B6_ Heavy _ chain CDRs\

CDR-H1-SEQ ID NO.15: TYAMS

CDR-H2-SEQ ID NO.16:

ISSRGRIYYPDSVKG CDR-H3-SEQ ID

NO.17: DYGSSYDY

4B6_ Light chain CDRs

CDR-L1-SEQ ID NO.18: RASQDIGSSLN

CDR-L2-SEQ ID NO.19: ATSSLDS CDR-L3-SEQ ID NO.20; LQYASSPWT 4G9_ Heavy _ chain CDRs :

CDR-H1-SEQ ID N0.21 :

RSSQSIVHSNGNTYLE CDR-H1-SEQ

ID N0.22: TVSNRFS

CDR-H1-SEQ ID N0.23: FQGSHVPPT

4G9 Light _ chain CDRs :

CDR-L1-SEQ ID N0.24: DYEMH

CDR-L1-SEQ ID N0.25:

AIDPGTGGIAYNQKFKG CDR-L1-SEQ

ID N0.26: YDYDLAY

Example 3:

Lenti viral - induced IL1RAP expression in KG1 cell line

Methods:

a. Construction of pHRST ILlRAP lentiviral vector

The lentiviral vector HRST-cmvGFP , was a kind gift from Richard C. Mulligan in the Harvard Gene Therapy Institute. IL1RAP cDNA was PCR amplified from Human IL- lRAcP/IL- iR3 Gene ORF cDNA clone expression plasmid(Sino biological INC, HG10121- CM) using Clone Amp HiFi PCR Premix(Takara Bio USA, Inc.), using primers including 5’ BamHI and Xhol sites;

F-IL1RAP

acgggatccccaccaagcttggtaccat

gac R-IL1RAP

acgctcgagttatacatttttcaaagatg

The PCR fragment was gel extracted as above, sub-cloned into BamHI and Xhol pHRST- MPSV vector according to standard protocols and confirmed by restriction mapping and sequencing.

b. Viral production (HEK transfection)

Transient production in adherent HEK293T was modified previously described(29). Briefly, HEK293T cells were seeded into T25 flasks to reach 70-80% confluence after day culture. Transfection was performed by adding a volume of 2.5M CaCl2(Sigma Aldrich, INC) to the plasmid DNA solution. The lentiviral transfer vector pHRST_ILlRAP DNA, together with psPAX2 packaging and pCMV-VSV- G envelope plasmid DNA were combined at a ratio of 13:5:2, respectively.

c.KGl lentiviral transduction with pHRST ILl RAP

Lentiviral transduction was done as previously described (30). In brief, lentivirus supernatant was added to KG1 cells cultured in complete IMEM. After overnight incubation, lentivirus was removed and fresh media added. Following 48 hrs (initial culturing conditions) the cells

were removed and IL1RAP+ cells were determined by flow cytometry using anti- Human IL-l RAcP/IL-l R3 PE-conjugated Antibody (#FAB676P, R and D, INC)

Results:

FIG.5 shows a histogram illustrating the level of CD 176 expression in KG1 cell line before and after VCN while FIG.6 shows how IL1RAP was induced as a consequence of lentiviral expression of KG1 cells (KG1/RAP; a cell line positive for both antigens). A. pHRST- MPSV/CMV and pHRST-ILlRAP. B. Construction of pHRST-ILlRAP lentiviral vector and verification. The 700 bp GFP band was digested using BamHI/XhoI restriction enzymes. IL1RAP cDNA was PCR amplified with primers flanked with

BamHI/XhoI sequences then ligated to the linearized pHRST-MPSV lentiviral backbone, transformed into E-coli and screened for colonies. Colonies were screened using HINDIII

, and an extra 1KB band (red box) was diagnostic for pHRST-MPSV-ILlRAP construct. V ;Vector, M: DNA marker, I; insert, PC: positive control. C. Histogram showing IL1RAP expression in KG1/RAP versus KG1 cell line. D. IL1RAP expression was increased by more than 50% as a result of lentiviral transduction. Each point represents Mean ± SEM of the four samples.

Example 4: Bί-specific antibody validation in cell lines and human samples.

Methods:

HEK 293T/17 cells (ATCC # CRL- 11268) were cultured in DMEM ( Life technologies, INQsupplemented with 10% heat-inactivated fetal bovine serum (FBS) , 100 U/ml penicillin, 100 pg/ml streptomycin sulfate, and 4.0 mM L-glutamine (Gibco BRL products, Gaithersburg, MD), at 37°C in a humidified 5% C02 incubator. KG1 cell line (ATCC #CCL-246) were cultured in Iscove's Modified Dulbecco's Medium (Life technologies, INC) supplemented with a fetal bovine serum to a final concentration of 20% prior to transduction.

Monoclonal MouselgGl clone # 89412 (#FAB676P, R and D systems), Primary APC Mouse Anti-Human IgG Clone G18-145 (RUO), Mouse IgGl, <#550931, BD Pharmingen™ ), Primary LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation(#L34957, Invitrogen ), VCN (Vibrio Cholera Neuraminidase) (Sigma Aldrich Inc), an enzyme used to expose the CD 176 on the surface of expressing cells.

Complement-dependent cytotoxicity (CDC)

The capacity to induce CDC was assessed essentially as described (30). Briefly, target cells (1 x 105 cells) were pre-incubated at 37 °C for 60 min with serially diluted antibodies.

Human Serum from human male AB (Sigma Aldrich) (20% (v/v)) was added as a source of complement and cells were incubated at 37 °C for an additional 45 min. Cells were then put on ice and viability was determined by staining with LIVE/DEAD™ staining and detected

using a FORTESSA flow cytometer (BD Biosciences). CDC activity was expressed as a percentage of lyses as determined from the percentage of LIVE/DEAD™ -positive cells. Cycloviolacin 02 (CyO2)(0.05nM); a positive control that kills cells with the same mechanism as CDC; causes potent cell membrane disruption.

Statistics

For each experiment, more than three replicated were conducted and the results were expressed as average ± standard deviation. Comparison of multiple groups was conducted using ANOVA based Test and p < 0.05 (*) represented significances with statistical meaning. Results:

CD 176 antigen is a glycosylated antigen; a protein antigen bound to GAL-NAC moiety which makes the antigen hidden on the cell surface and not easy to isolate (Ju et al.,20l4). For this reason, Flow Cytometry assay was used in order to evaluate both the binding affinity and toxicity of our bi-specific without the need to purify the antigen or even knowing the antigen identity FIG.7 shows the gating strategy used for analysis of the bi-specific antibody binding and killing. Binding ability was calculated from the number of IgG positive cells, where the secondary IgG antibody is bound to the primary Bis-Ab. The four cocktails were first evaluated in KG1/KG1/RAP cell lines then in CML samples.TFlRAPa Bis-Ab showed the highest binding affinity in KG1/RAP cells (Figure 8A) as compared to other cocktails (pO.OOl). It also showed the highest binding in terms of percentages of positive IgG events(data not shown). In contrast, the third antibody TFlRAPb revealed the least binding ability in KG1/RAP cells. Subsequently, the other three cocktails; TFlRAPa, TF2RAPa, and TF2RAPc were tested in CML PBMCs, and again TFlRAPa has shown the maximum receptor saturation as compared to other cocktails (Figure 8D).

Therapeutic antibodies, such as ofatumumab directed against CD20, have shown significant CDC against peripheral blood cells obtained from CML patients in chronic phases (Tatake et al.,1990) and B cells in CLL (Margaret et al., 2013) respectively. Thus, increasing doses of TFlRAPa- containing, HEK supernatant, were used to generate a pre- liminary dose-response curve, to test whether CDC could be achieved using IL1RAP and CD176 as targets. The ability of TFlRAPa and a control IgG antibody to induce cell death was investigated in duel positive cell lines versus CML PBMCs. In the IL1RAP- leukemia cell line KG1, lacking IL1RAP expression (Fig. 8B), only a low level of binding and CDC were observed, even at high TFlRAPa concentrations (Fig. 8C). In contrast, in the IL1RAP+ leukemia cell line KG 1 /RAP, expressing both CD 176 IL1RAP antigens (Fig. 8B), relatively high CDC was observed in the presence of TFlRAPa (Fig. 8C), demonstrating that TFlRAPa has the potential to induce CDC by binding IL1RAP and CD 176

antigens on leukemic target cells. PBMCs from CML1-4 were tested in CDC assays in parallel to cells from healthy control samples. In CML cells, the binding of TFIRAPa mediated CDC at higher levels than in normal peripheral blood mononuclear control cells, correlating with the expression level of IL1RAP and CD 176, in particular at lower antibody concentrations (FIG. 8E- F). To address the selectivity of IL1 RAP/CD 176- targeting antibodies, we also validated the bi- specific antibody cytotoxicity on various cell populations of peripheral blood. CD176+IL1RAP+ cell populations have shown the highest CDC effect as compared to other populations; CD176+IL1RAP-, CD 176- IL1RAP+, and CD176-IL1RAP- (FIG.9, FIG.10A-C).

FIG.8 provides how the bi-specific antibody was validated in cell lines and CML samples. A. Bar graph showing different binding abilities for the four TF-RAP cocktails in KG1 and KG 1 /RAP cell lines. A significant difference was observed between the four antibodies in terms of binding to KG1/RAP cell line (p < 0.01). TFlRAPa showed the highest mean expression of IgG in KG1/RAP cell line. KG1 cell line is devoid of IL1RAP expression, whereas KG1/RAP cells express both IL1RAP and CD176. As a consequence, only a low level of antibody-binding was observed in KG1 (B), but a dose- dependent CDC effect was observed using TFlRAPa on KG1/RAP cells (C). The binding capacity was depicted from the percentage of IgG bound cells.

D. Receptors saturation of increasing concentrations of TFlRAPa in CML PBMCs was assessed. TFlRAPa antibody is showing a higher affinity to CD176+IL1RAP+ receptors, as compared to the CD176+IL1RAP- and CD176-IL1RAP- ones. The amount of bi specific antibody bound to the receptors was calculated from the following equation ((Frequency of total IgG bound receptors/Freq of total free receptors)* 100). CML samples were treated with increasing doses of bi-specific antibody- transfected HEK supernatant for one hour, and increasing binding and killing were observed (p < 0.01) (E, F). 10²-10⁴ cells were analyzed per replicate. Each point represents the Mean ± SEM of three to four replicates.

FIG.9 shows different receptors interaction with TFlRAPa, as compared to CD176+IL1RAP+. Using Post-Floc Bonferroni test, significant difference was observed on comparing between CD176+IL1RAP+ to CD176+IL1RAP- and CD176-IL1RAP- and not CD 176-IL 1 RAP+. FIG.10 shows a dose-response curve demonstrating the selective killing potential of the TFIRAPa to CD176+IL1RAP+ as compared to other subpopulations (p < 0.05). Each point represents the Mean ± SEM of the four samples.

More strikingly, among peripheral blood cells, TFIRAPa did not induce CDC of normal cells, whereas a clear dose-dependent CDC effect was observed in CML. FIG.11 shows the killing of CML PBMCs CDl76+ILiRAP+cells by the bi-specific antibody; TFlRAPa. In line with the level of CD 176 and IL1RAP expression, no obvious CDC effect was seen in normal PBMCs (A) versus CML (B) cells, whereas TFIRAPa induced the strongest dose-dependent CDC effect in CML CD176+IL1RAP+ (C) as compared to other populations (D-F). As a control for nonspecific CDC effects, a mouse anti-human IgG antibody was also used in the experiments. Each graph shows the average and SD of antibody-induced cell death from a minimum of three CML samples.

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BRIEF DESCRIPTION OF THE DRAWINGS:

FIG. l shows the mechanisms of action of Bis-Abs.

FIG.2 showing how CD176 and IL1RAP antigens are co-expressed on CD34⁺Leukemia stem cells. FIG.3. shows FISH analysis of CD34+CDl76+ILlRAP+population in CML-4.

FIG .4 schematically illustrates the four bi-specific antibody arms.

FIG.5 A histogram illustrating the level of CD 176 expression in KG1 cell line before and after VCN

FIG.6 showing IL1RAP is induced as a consequence of lentiviral expression of KG1 cells. FIG.7 shows the gating strategy used for analysis of the bi-specific antibody binding and killing.

FIG.8 provides how the bi-specific antibody was validated in cell lines and CML samples.

FIG.9 shows the killing of CML PBMCs CDl76+ILlRAP+cells by the bi-specific antibody; TFlRAPa.

FIG.10 shows a dose-response curve demonstrating the selective killing potential of the TFlRAPa to CD 176+IL 1 RAP+ as compared to other subpopulations.

FIG.l 1 shows different receptors interaction with TFlRAPa, as compared to CD176+IL1RAP+.

Claims

Claims:

1. A method of preparing a bi-specific antibody, having at least one arm that specifically binds a targeted oncoprotein; and at least one other arm that specifically binds a membranous protein comprises integrating two approaches: 1) orthogonal Fab platform previously used by Lewis et al.,2014) and 2)Knob in hole strategy, where four different antibody arms were generated. Arm one comprises the variable domain sequence of the heavy chain of one antigen followed by an orthogonal Fab sequence of the heavy chain of the first constant domain of human IgG followed by a knob in a hole sequence in the heavy of the third constant domain. Arm two comprises the variable domain sequence of the light chain of same previous antigen followed by an orthogonal Fab sequence of the light chain of the constant domain of human IgG. The third arm however comprises the variable domain sequence of the heavy chain of the second antigen followed by the sequence of the heavy chain of the first constant domain of the wild type human IgG followed by a knob in a hole sequence in the heavy of the third constant domain. And finally the fourth arm comprises the variable domain sequence of the light chain of the second antigen followed by the wild type sequence of the light chain of the constant domain of human IgG. Two different clones for each, variable domain i.e. for each antigen were used alternatively.

2. The method of claim 1, wherein said at least one arm that specifically targeted oncoprotein; and at least one other arm that specifically binds a membranous protein is a human Fab antibody.

3. The method of claim 1, wherein said at least one other arm specifically binds said epitope of said targetable oncoprotein, and said epitope comprises Thosmen Freidenrich(CDl76) antigen, SEQID N0.9-14.

4. The method of claim 1, wherein said at least one other arm specifically binds said epitope of said targetable membranous protein, and said epitope is interleukin- 1 accessory protein(ILl RAPP), SEQID NO.15-26 .

5.The method of claim 1, wherein said, orthogonal fab platform, and said platform comprises two sequences, CH1 H172AF174G, CL_Ll35Y_Sl76W.

6. The method of claim 1, wherein said, knob in hole, and said comprises two sequences; T366W :S354C.

7.The method of claim 1, wherein said arm one comprises the sequence of variable domain sequence of the heavy chain of one antigen followed by an orthogonal Fab sequence of the heavy chain of the first constant domain of human IgG followed by a knob in a hole sequence in the heavy of the third constant domain and said sequence is SEQ ID NO.l and SEQ ID NO.5.

8.The method of claim 1, wherein said arm two comprises the variable domain sequence of the light chain of same previous antigen followed by an orthogonal Fab sequence of the light chain of the constant domain of human IgG and said sequence is SEQ ID NO.2 and SEQ ID NO.6.

9. The method of claim 1 , wherein said the third arm however comprises the sequence of the variable domain sequence of the heavy chain of the second antigen followed by the sequence of the heavy chain of the first constant domain of the wild type human IgG followed by a knob in a hole sequence in the heavy of the third constant domain, and said sequence is SEQ ID NO.3 and SEQ ID NO.7

10. The method of claim 1, wherein said the fourth arm comprises the variable domain sequence of the light chain of the second antigen followed by the wild type sequence of the light chain of the constant domain of human IgG, and said sequence is SEQ ID NO.4 and SEQ ID NO.8.

11. The peptide sequence in claim 5 wherein said, and said platform comprises two sequences, CHl_Hl72AFl74G, CL_Ll35Y_Sl76W, sequence CHl_H172AFl74G was part of SEQ ID.landSEQ

ID.5.

12. The peptide sequence in claim 5 wherein said, and said platform comprises two sequences, CHl_Hl72AFl74G, CL_Ll35Y_S176W, CL_Ll35Y_Sl76W was part of SEQ ID.2 and SEQ ID.6.

13. The peptide sequence in claim 6 wherein said, knob in hole, and said comprises two sequences; T366W:S354C, T366W was part of SEQ ID.l and SEQ

ID.5.

14. The peptide sequence in claim 6 wherein said, knob in hole, and said comprises two sequences; T366W:S354C, S354C was part of SEQ ID.3 and SEQ ID.7.

15. The antibody mentioned in claim 1 wherein said, a bi-specific antibody, having at least one arm that specifically binds a targeted oncoprotein; and at least one other arm that specifically binds a membranous protein, kills cancer cells by complement dependent cytotoxicity(CDC).