NOVEL AGENTS AND USES THEREOF
20230092356 · 2023-03-23
Inventors
Cpc classification
International classification
Abstract
The present invention provides agents comprising or consisting of a binding moiety with specificity for Signaling Lymphocytic Activating Molecule Family Member 6 (SLAMF6) for use in inducing cell death and/or inhibiting the growth and/or proliferation of pathological stem cells and/or progenitor cells associated with a neoplastic hematologic disorder, wherein the cells express SLAMF6 and/or modulating their interactions with immune cells that may also express SLAMF6. A related aspect of the invention provides agents comprising or consisting of a binding moiety with specificity for SLAMF6 for use in detecting pathological stem cells, progenitor cells and/or immune cells associated with a neoplastic hematologic disorder, wherein the cells express SLAMF6. Further provided are pharmacological compositions comprising the agents of the invention and methods of using the same.
Claims
1. A method for: (i) inducing cell death of pathological stem cells and/or progenitor cells associated with a neoplastic hematologic disorder; and/or (ii) inhibiting the growth and/or proliferation of pathological stem cells and/or progenitor cells associated with a neoplastic hematologic disorder; or (iii) detecting pathological stem cells and/or progenitor cells associated with a neoplastic hematologic disorder, wherein the cells express Signaling Lymphocytic Activating Molecule Family Member 6 (SLAMF6), and wherein the method comprises administering an agent comprising or consisting of a binding moiety with specificity for SLAMF6.
2. (canceled)
3. A method according to claim 1, wherein the neoplastic hematologic disorder: (a) is a leukemia; (b) is associated with cells comprising a TP53 mutation; and/or (c) is selected from the group consisting of chronic myeloid leukemia (CML), myeloproliferative disorders (MPD), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), preferably acute myeloid leukemia (AML).
4. A method according to claim 1, wherein the cells expressing SLAMF6 also express CD34.sup.+CD38.sup.−, optionally wherein the cells comprise a TP53 mutation.
5. A method according to claim 1, wherein the binding moiety has specificity for human SLAMF6, and/or wherein SLAMF6 is localised on the surface of a cell.
6. A method according to claim 1, wherein the agent is capable of modulating an interaction between an immune cell and leukemic cells and/or an immune cell and leukemic stem cells, optionally wherein the immune cells are selected from the group consisting of: B cells, T cells and/or NK cells; preferably wherein the immune cells express SLAMF6.
7. A method according to claim 1, wherein the agent: (a) is capable of killing the pathological stem cells and/or progenitor cells, optionally by antibody-dependent cell-mediated cytotoxicity (ADCC) and/or by a T cell mediated mechanism; (b) is capable of inducing apoptosis of the stem cells and/or progenitor cells, optionally by antibody-dependent cell-mediated cytotoxicity (ADCC) and/or by a T cell mediated mechanism.
8. A method according to claim 1, wherein the agent comprises or consists of: (a) a polypeptide; and/or (b) an antibody or an antigen-binding fragment thereof with binding specificity for SLAMF6, or a variant, fusion or derivative of said antibody or antigen-binding fragment, or a fusion of a said variant or derivative thereof, which retains the binding specificity for SLAMF6; and/or (c) an antibody or antigen-binding fragment thereof with binding specificity for SLAMF6, optionally wherein the agent comprises or consists of an intact antibody or an antigen-binding fragment of an antibody, such as an antigen-binding fragment selected from the group consisting of Fv fragments (e.g. single chain Fv, disulphide-bonded Fv and domain antibodies) and Fab-like fragments (e.g. Fab fragments, Fab′ fragments and F(ab).sub.2 fragments); and/or (d) a recombinant antibody; or (e) a monoclonal antibody; or (f) a polyclonal antibody; optionally wherein the antibody or antigen-binding fragment thereof is human or humanised; or (g) a non-immunoglobulin binding moiety; and/or (h) an aptamer, such as a peptide aptamer or a nucleic acid aptamer; or (i) a small chemical entity.
9. A method according to claim 1, wherein the agent further comprises: (a) a moiety for increasing the in vivo half-life of the agent, optionally wherein the moiety for increasing the in vivo half-life is selected from the group consisting of polyethylene glycol (PEG), human serum albumin, glycosylation groups, fatty acids and dextran; and/or wherein the agent is PEGylated; and/or (b) a cytotoxic moiety, optionally wherein the cytotoxic moiety comprises or consists of a radioisotope, such as a radioisotope selected from the group consisting of astatine-211, bismuth-212, bismuth-213, iodine-131, yttrium-90, lutetium-177, samarium-153 and palladium-109; or wherein the cytotoxic moiety comprises or consists of a toxin (such as saporin or calicheamicin) or a chemotherapeutic agent (such as an antimetabolite); and/or (c) a detectable moiety, optionally wherein the detectable moiety comprises or consists of a radioisotope, such as a radioisotope selected from the group consisting of: technetium-99m; indium-111; gallium-67; gallium-68; arsenic-72; zirconium-89; iodine-12; thallium-201; or wherein the detectable moiety comprises or consists of a paramagnetic isotope, such as a paramagnetic isotope selected from the group consisting of: gadolinium-157; manganese-55, dysprosium-162, chromium-52; iron-56.
10. A pharmaceutical composition comprising an effective amount of an agent comprising or consisting of a binding moiety with specificity for SLAMF6 and a pharmaceutically-acceptable diluent, carrier or excipient, optionally adapted for parenteral delivery or intravenous delivery.
11-12. (canceled)
13. A method according to claim 1, wherein the method is for inducing cell death and/or inhibiting the growth and/or proliferation of pathological stem cells and/or progenitor cells associated with a neoplastic hematologic disorder in an individual, comprising the step of administering to the individual an effective amount of an agent comprising or consisting of a binding moiety with specificity for SLAMF6, wherein the cells express SLAMF6.
14. A method according to claim 1, wherein the method is for detecting pathological stem cells and/or progenitor cells associated with neoplastic hematologic disorder in an individual and comprises the step of administering to the individual an effective amount of an agent comprising or consisting of a binding moiety with specificity for SLAMF6, wherein the cells express SLAMF6.
15. An in vitro method for diagnosing or prognosing a neoplastic hematologic disorder, the method comprising: (a) providing a bone marrow or peripheral blood sample of haematopoietic cells from an individual to be tested; (b) isolating a subpopulation of CD34.sup.+, CD38.sup.− cells from the haematopoietic cells; and (c) determining whether stem cells, contained within the CD34.sup.+, CD38.sup.− cells, express the cell surface marker SLAMF6; wherein stem cells that exhibit the cell surface marker profile CD34.sup.+, CD38.sup.− and SLAMF6.sup.+ are indicative of the individual having or developing leukemia; optionally wherein the method also includes a step comprising quantification of levels of immune cells (such as B cells, T cells and/or NK cells), preferably wherein the immune cells express SLAMF6.
16. A method according to claim 1, wherein the neoplastic hematologic disorder is a leukemia, such as a neoplastic hematologic disorder selected from the group consisting of chronic myeloid leukemia (CML), myeloproliferative disorders (MPD), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), preferably acute myeloid leukemia (AML).
17. (canceled)
Description
[0133] Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figures:
[0134]
[0135] A: Gates used to define immature, viable, single cells with a CD3.sup.−CD19.sup.−CD34.sup.+CD38.sup.− phenotype. B: Waterfall plot of median difference in MFI between AML and NBM within the CD3.sup.−CD19.sup.−CD34.sup.+CD38.sup.− compartment. Three TP53 mutated AML and three NBM samples were analyzed. Markers with high expression in NBM are excluded from this plot. C: Spearman correlation of MFI between biological replicates from antibody screen for NBM #2 and NBM #3. 14 values not depicted due to value of zero or negative values and logarithmic axes. D: Spearman correlation of MFI between biological replicates from antibody screen for AML #83 and AML #80. 42 values not depicted due to value of zero or negative values. E: Top ranked cell surface markers based on difference in AML .sup.MFI and NBM.sup.MFI from three separate analyses of TP53 mutated AML and NBM samples. Median MFI is plotted for AML (black bars) and NBM (white bars).
[0136]
[0137] Eight novel cell surface receptors with high ranking in the screen were analyzed in separate experiments. Representative histograms of an AML (far left), an NBM sample (middle left), percent positive cells (middle right), and MFI (far right) for each marker within the CD34.sup.+CD38.sup.− population are shown. Mean and standard deviation are shown.
[0138]
[0139] A: AML SLAMF6 expression is higher in phenotypically immature CD34.sup.+CD38.sup.+ and CD34.sup.+CD38.sup.− populations. Histograms from a representative sample (AML 48) shown. B: NBM SLAMF6 expression is detected in mature CD34.sup.−CD38.sup.− and CD34.sup.−CD38.sup.+ NBM population but low or absent in more immature CD34.sup.+CD38.sup.+ and CD34.sup.+CD38.sup.− respectively. Histograms from one representative sample shown. C: SLAMF6 gene expression levels are higher in TP53 mutated AML MNC cells than in stem and progenitor populations of normal bone marrow. CMP: common myeloid progenitor, GMP: granulocyte-macrophage progenitor, MEP: megakaryocyte-erythroid progenitor, LMPP: lymphoid-primed multipotent progenitor, MPP: multipotent progenitor, HSC: hematopoietic stem cells. D: CD3.sup.+ T cells and CD19.sup.+ B cells in AML samples retain their SLAMF6 expression in all tested genetic subgroups of AML. AML 66 is shown as a representative example.
[0140]
[0141] A: SLAMF6 gene expression levels are significantly higher in TP53 mutated AML samples (n=14) compared to AML samples of other genetic subtypes (n=151) in the publicly available TCGA dataset. B: SLAMF6 is expressed in the leukemic CD19.sup.−CD3.sup.− population in a TP53 mutated CD34 negative AML sample. C: SLAMF6 is expressed on leukemic cells in AML 33, AML 34 and on a subpopulation of MDS144. D: SLAMF6 is possibly expressed at low levels on leukemic cells in AML 32, MDS35 and AML 66. E: No expression of SLAMF6 could be detected on the leukemic cells in AML 21, AML 28, MDS70 or AML 94.
[0142]
[0143] A: Histograms showing SLAM6 expression in the AML cell lines KG1 and K562 but not OCI-AML 3. B: Specific SLAMF6 mediated ADCC killing of KG1 cells (black, solid line) compared to isotype control (grey, dashed line). Mean effect and standard deviation of three different NK donors are shown, results are normalization to base line killing by the NK cells. C: Xenografts of TP53 mutated AML show high levels of SLAMF6 expression within the human CD45.sup.+CD34.sup.+CD38.sup.− compartment. Spleen from secondary an AML 48 xenograft is shown as a representative example with 99% CD45.sup.+ human cells, 45% CD34.sup.+CD38.sup.− cells and a high SLAMF6 expression.
[0144]
[0145] A. Specific killing of AML patient cells ex vivo by a SLAMF6 antibody eliciting NK cell recruitment and killing through ADCC. Values indicate the average number of remaining viable target cells+/−SEM after treatment with a SLAMF6 antibody (black, solid line) or an isotype control (grey, dashed line), normalized to a control without antibody. For AML-83, cells were passaged in vivo for two generations for LSC enrichment before being subjected to ADCC. One NK cell donor was used for PDX-83 and four for AML-61. B. Protein expression of SLAMF6 on the AML patient cells.
[0146]
[0147] SLAMF6 as determined by flow cytometry is expressed on a majority of AML samples, in particular in the CD34+CD38low leukemic stem cell containing fraction. Samples were classified as “High” when containing >50% SLAMF6 positive cells, “Intermediate” when containing 10-50% SLAMF6 positive cells and “Negative” when containing <10% SLAMF6 positive cells.
[0148]
[0149] SLAMF6 as determined by flow cytometry is expressed on leukemic stem cells from AML samples with a large variety of mutational backgrounds.
[0150]
[0151] SLAMF6 as determined by flow cytometry is expressed on the AML cell lines KG1 (DSMZ #ACC14), HNT-34 (DSMZ #ACC600), TF-1 (DSMZ #ACC334), CMK (DSMZ #ACC392), and K562 (DSMZ #ACC10) but not on THP-1 (DSMZ #ACC16), OCI-AML 3 (DSMZ #ACC582), NB4 (DSMZ #ACC207), or MonoMac6 (DSMZ #ACC124).
[0152]
[0153] T cell-mediated killing of HNT-34 cells, measured as the total number of target cells 72 h after seeding of T cells and HNT-34 target cells at a 4:1 ratio, with the addition of an activating SLAMF6 antibody or an isotype control.
[0154]
[0155] T cell-mediated killing of SLAMF6 knockout cells, measured as the total number of target cells (A), T cells (B) and activated T cells (C) three days after seeding of T cells and KG-1 target cells at a 4:1 ratio. Dashed line indicates the number of cells seeded. (D) Validation of SLAMF6 knockout by FACS.
EXAMPLE 1
[0156] SLAMF6 is a Cell Surface Biomarker for Acute Myeloid Leukemia Stem Cells
[0157] Summary
[0158] Therapeutic strategies for acute myeloid leukemia (AML) aiming at achieving a permanent cure of the disorder, will require a full eradication of the AML stem cells. The AML stem cells, sharing the capacity to self-renew with normal hematopoietic stem cells (HSCs), represent a small population of leukemic cells that so far have been indistinguishable from normal (HSCs) using cell surface markers. One strategy to target the AML stem cell would be to identify a cell surface biomarker for AML stem cells, to which future therapeutic antibodies could be directed. In this study, SLAMF6 was identified in a surface marker screen of TP53-mutated AML as commonly expressed on primitive CD34.sup.+CD38.sup.− AML cells but not on corresponding normal cells. Furthermore, targeting of SLAMF6 was shown to induce killing of AML cells through antibody-dependent cellular cytotoxicity (ADCC).
[0159] This study thus identifies SLAMF6 as a novel cell surface biomarker distinguishing AML stem cells from normal HSC and opens up new avenues for therapeutic and diagnostic strategies in AML. In addition to direct effects on the leukemia cell, targeting of SLAMF6 may also alter its interaction with immune cells to induce an anti-tumor response. Further, as SLAMF6 is also expressed on immune cells (B cells, T cells and NK cells), it is a suitable target for dual targeting on both pathological stem cells (e.g. leukemic stem cells) and immune cells (e.g. B cells, T cells and/or NK cells), whereby the immune cells may be activated to enhance elimination of the pathological stem cells. For example, the agent may be used to target pathological stem cells and B cells; or pathological stem cells, B cells and T cells; or pathological stem cells, NK cells and T cells, etc.
[0160] Introduction
[0161] To identify a cell surface biomarker for AML stem cells, the inventors performed an antibody screen and identified SLAMF6 as a novel candidate, being upregulated in primitive AML patient cells.
[0162] Materials and Methods
[0163] Patient Samples
[0164] Bone marrow and peripheral blood samples were collected after written informed consent in accordance to the Declaration of Helsinki. Samples were collected from patients with AML, myelodysplastic syndrome (MDS) or healthy controls. Mononuclear cells (MNC) were isolated using Lymphoprep (GE Healthcare Bio-Sciences AB, Sweden) and subsequently viably frozen. Patients included in the study and their clinical characteristics are shown in Table 1. The study was approved by a regional ethics committee in Lund (Dnr2011/289).
[0165] Cell Surface Marker Screen
[0166] Arrayed antibody libraries were prepared based on the LEGENDScreen system (BioLegend, USA), containing 362 PE-conjugated antibodies. Two different iterations of the LEGENDScreen (BioLegend, USA) were used, containing slightly different antibodies (#700001 had 34 antibodies not included in #700007 which contained 61 antibodies not included in #700001; for a complete list, see Table 2). Antibodies targeting IL1RAP, previously shown to be upregulated on AML stem cells,.sup.9, 22, 23 and CD177, suggested to be upregulated on the mRNA level in AML (data not shown), were added to the arrays. Antibodies against CD3, CD19, CD34, CD38 and the viability marker 7AAD were added to each well of the 96-well U-shaped plates. All antibodies and reagents used are listed in Table 3. Bone marrow mononuclear cells from TP53 mutated AML samples (n=3) or healthy normal bone marrow (NBM) donors (n=3) were added and incubated at 4° C. for 20 minutes and subsequently washed and resuspended. Flow cytometry read out was performed using an LSR Fortessa with an HTS (BD Bioscience, USA). Median fluorescent intensity (MFI) for each marker within the 7AAD.sup.−CD3.sup.−CD19.sup.−CD34.sup.+CD38.sup.− fraction were used to compare expression between AML and NBM (
TABLE-US-00001 TABLE 1 AML Patient Characteristics SLAMF6 in CD34+ Pat# Gender Disease FAB ELN Karyotype Mutations CD34 SLAMF6 CD38− 21 M AML-D M2 IR 46, XY IDH2, STAG2, BCOR pos neg neg 28 M SAML-D M2 IR 46, XY NPM1, FLT3-ITD, TET2 neg neg — 32 M AML-D M2 HR 47, XY, +13 RUNX1, ASXL1, pos low low 33 F SAML-R M4 IR 46, XX DNMT3A, NPM1, FLT3- neg pos — ITD, IDH1, CEBPA 34 F AML-D M5 IR 46, XX DNMT3A, IDH1, NRAS, pos pos pos CEBPA 35 F MDS — — 46, XX RUNX1, ASXL1, pos low low RAEB-2 DNMT3A, IDH2 48 F tAML-D M2 HR Complex1 TP53 pos pos pos 66 M SAML-D M2 LR 46, XY IDH1, NPM1, JAK2, neg low — CEBPA 70 M MPN/MDS — — 46, XY RUNX1, ASXL1, TET2, neg neg — 80 M AML-D M2 HR Complex2 TP53 pos pos pos 83 M SAML-D M2 HR Complex3 TP53, TET2, NF1 pos pos pos 94 M AML-D M0 HR 47, XY, +10 DNMT3A, IDH1, FLT3- pos neg neg ITD, RUNX1, BCOR, STAG2 144 F MPN/MDS — — 47, XX, +8 RUNX1, ASXL1, TET2, neg pos — BCOR 155 M AML-D M5 HR Complex4 TP53, FLT3 neg pos — Abbreviations: D: diagnosis, R: relapse, sAML: secondary AML, tAML: therapy-related AML, ELN: European Leukemia net risk classification 2017, IR: intermediate risk, HR: high risk, CD34 pos: ≥15% CD34+ cells Complex1: 43-47, XX, del(5)(q13q33), der(8)t(8;12)(p22;p13), add(11)(p15), −12, add(13)(p11), −18, del(20)(q11), add(22)(q13), +mar Complex2: 48, XXYc, +i(12)(p10), der(17)t(13;17)(q1?4;p1?3) Complex3: 45, XY, der(13;14)(q10;q10)/42-44, idem, −5, −7, −9, −10, −11, ?hsr(11)(q23), −14, −16, −20, +4mar Complex4: 47, XY, +8, t(9;11)(p21;q23)/46-47, idem, der(9)del(9)(p12)del(9)(q12), der(17)t(9;17)(q31;p13)/46-47, idem, der(7;10)(q10;q10), +add(8)(p11)
TABLE-US-00002 TABLE 2 Antibodies included in the screen Screen Iteration 70001 Plate Well ID Specificity Clone Isotype Cat# Plate 1 A01 Blank Plate 1 A02 CD1a HI149 Mouse IgG1, κ 300106 Plate 1 A03 CD1b SN13 (K5- 1B8) Mouse IgG1, κ 329108 Plate 1 A04 CD1c L161 Mouse IgG1, κ 331506 Plate 1 A05 CD1d 51.1 Mouse IgG2b, κ 350306 Plate 1 A06 CD2 RPA-2.10 Mouse IgG1, κ 300208 Plate 1 A07 CD3 HIT3a Mouse IgG2a, κ 300308 Plate 1 A08 CD4 RPA-T4 Mouse IgG1, κ 300508 Plate 1 A09 CD5 UCHT2 Mouse IgG1, κ 300608 Plate 1 A10 CD6 BL-CD6 Mouse IgG1, κ 313906 Plate 1 A11 CD7 CD7-6B7 Mouse IgG2a, κ 343106 Plate 1 A12 CD8a HIT8a Mouse IgG1, κ 300908 Plate 1 B01 CD9 HI9a Mouse IgG1, κ 312106 Plate 1 B02 CD10 HI10a Mouse IgG1, κ 312204 Plate 1 B03 CD11a HI111 Mouse IgG1, κ 301208 Plate 1 B04 CD11b ICRF44 Mouse IgG1, κ 301306 Plate 1 B05 CD11b (activated) CBRM1/5 Mouse IgG1, κ 301406 Plate 1 B06 CD11c 3.9 Mouse IgG1, κ 301606 Plate 1 B07 CD13 WM15 Mouse IgG1, κ 301704 Plate 1 B08 CD14 M5E2 Mouse IgG2a, κ 301806 Plate 1 B09 CD15 (SSEA-1) W6D3 Mouse IgG1, κ 323006 Plate 1 B10 CD16 3G8 Mouse IgG1, κ 302008 Plate 1 B11 CD18 TS1/18 Mouse IgG1, κ 302108 Plate 1 B12 CD19 HIB19 Mouse IgG1, κ 302208 Plate 1 C01 CD20 2H7 Mouse IgG2b, κ 302306 Plate 1 C02 CD21 Bu32 Mouse IgG1, κ 354904 Plate 1 C03 CD22 HIB22 Mouse IgG1, κ 302506 Plate 1 C04 CD23 EBVCS-5 Mouse IgG1, κ 338508 Plate 1 C05 CD24 ML5 Mouse IgG2a, κ 311106 Plate 1 C06 CD25 BC96 Mouse IgG1, κ 302606 Plate 1 C07 CD26 BA5b Mouse IgG2a, κ 302706 Plate 1 C08 CD27 O323 Mouse IgG1, κ 302808 Plate 1 C09 CD28 CD28.2 Mouse IgG1, κ 302908 Plate 1 C10 CD29 TS2/16 Mouse IgG1, κ 303004 Plate 1 C11 CD30 BY88 Mouse IgG1, κ 333906 Plate 1 C12 CD31 WM59 Mouse IgG1, κ 303106 Plate 1 D01 CD32 FUN-2 Mouse IgG2b, κ 303206 Plate 1 D02 CD33 WM53 Mouse IgG1, κ 303404 Plate 1 D03 CD34 581 Mouse IgG1, κ 343506 Plate 1 D04 CD35 E11 Mouse IgG1, κ 333406 Plate 1 D05 CD36 5-271 Mouse IgG2a, κ 336206 Plate 1 D06 CD38 HIT2 Mouse IgG1, κ 303506 Plate 1 D07 CD39 A1 Mouse IgG1, κ 328208 Plate 1 D08 CD40 HB14 Mouse IgG1, κ 313006 Plate 1 D09 CD41 HIP8 Mouse IgG1, κ 303706 Plate 1 D10 CD42b HIP1 Mouse IgG1, κ 303906 Plate 1 D11 CD43 CD43-10G7 Mouse IgG1, κ 343204 Plate 1 D12 CD44 BJ18 Mouse IgG1, κ 338808 Plate 1 E01 CD45 HI30 Mouse IgG1, κ 304008 Plate 1 E02 CD45RA HI100 Mouse IgG2b, κ 304108 Plate 1 E03 CD45RB MEM-55 Mouse IgG2b, κ 310204 Plate 1 E04 CD45RO UCHL1 Mouse IgG2a, κ 304206 Plate 1 E05 CD46 TRA-2-10 Mouse IgG1 352402 Plate 1 E06 CD47 CC2C6 Mouse IgG1, κ 323108 Plate 1 E07 CD48 BJ40 Mouse IgG1, κ 336708 Plate 1 E08 CD49a TS2/7 Mouse IgG1, κ 328304 Plate 1 E09 CD49c ASC-1 Mouse IgG1, κ 343804 Plate 1 E10 CD49d 9F10 Mouse IgG1, κ 304304 Plate 1 E11 CD49e NKI-SAM-1 Mouse IgG2b, κ 328010 Plate 1 E12 CD49f GoH3 Rat IgG2a, κ 313612 Plate 1 F01 CD50 (ICAM-3) CBR-IC3/1 Mouse IgG1, κ 330005 Plate 1 F02 CD51 NKI-M9 Mouse IgG2a, κ 327910 Plate 1 F03 CD51/61 23C6 Mouse IgG1, κ 304406 Plate 1 F04 CD52 HI186 Mouse IgG2b, κ 316006 Plate 1 F05 CD53 HI29 Mouse IgG1, κ 325406 Plate 1 F06 CD54 HA58 Mouse IgG1, κ 353106 Plate 1 F07 CD55 JS11 Mouse IgG1, κ 311308 Plate 1 F08 CD56 (NCAM) HCD56 Mouse IgG1, κ 318306 Plate 1 F09 CD57 HCD57 Mouse IgM, κ 322312 Plate 1 F10 CD58 TS2/9 Mouse IgG1, κ 330905 Plate 1 F11 CD59 p282(H19) Mouse IgG2a, κ 304708 Plate 1 F12 CD61 VI-PL2 Mouse IgG1, κ 336406 Plate 1 G01 CD62E HAE-1f Mouse IgG1, κ 336008 Plate 1 G02 CD62L DREG-56 Mouse IgG1, κ 304806 Plate 1 G03 CD62P (P-Selectin) AK4 Mouse IgG1, κ 304906 Plate 1 G04 CD63 H5C6 Mouse IgG1, κ 353004 Plate 1 G05 CD64 10.1 Mouse IgG1, κ 305008 Plate 1 G06 CD66a/c/e ASL-32 Mouse IgG2b, κ 342304 Plate 1 G07 CD66b G10F5 Mouse IgM, κ 305106 Plate 1 G08 CD69 FN50 Mouse IgG1, κ 310906 Plate 1 G09 CD70 113-16 Mouse IgG1, κ 355104 Plate 1 G10 CD71 CY1G4 Mouse IgG2a, κ 334106 Plate 1 G11 CD73 AD2 Mouse IgG1, κ 344004 Plate 1 G12 CD74 LN2 Mouse IgG1, κ 326808 Plate 1 H01 CD79b CB3-1 Mouse IgG1, κ 341404 Plate 1 H02 CD80 2D10 Mouse IgG1, κ 305208 Plate 1 H03 CD81 5A6 Mouse IgG1, κ 349506 Plate 1 H04 CD82 ASL-24 Mouse IgG1, κ 342104 Plate 1 H05 CD83 HB15e Mouse IgG1, κ 305308 Plate 1 H06 CD84 CD84.1.21 Mouse IgG2a, κ 326008 Plate 1 H07 CD85a (ILT5) MKT5.1 Rat IgG2a, κ 337704 Plate 1 H08 CD85d (ILT4) 42D1 Rat IgG2a, κ 338706 Plate 1 H09 CD85g (ILT7) 17G10.2 Mouse IgG1, κ 326408 Plate 1 H10 CD85h (ILT1) 24 Mouse IgG2b, κ 337904 Plate 1 H11 CD85j (ILT2) GHI/75 Mouse IgG2b, κ 333708 Plate 1 H12 CD85k (ILT3) ZM4.1 Mouse IgG1, κ 333008 Plate 2 A01 Blank Plate 2 A02 CD86 IT2.2 Mouse IgG2b, κ 305406 Plate 2 A03 CD87 VIM5 Mouse IgG1, κ 336906 Plate 2 A04 CD88 S5/1 Mouse IgG2a, κ 344304 Plate 2 A05 CD89 A59 Mouse IgG1, κ 354104 Plate 2 A06 CD90 (Thy1) 50000000000 Mouse IgG1, κ 328110 Plate 2 A07 CD93 VIMD2 Mouse IgG1, κ 336108 Plate 2 A08 CD94 DX22 Mouse IgG1, κ 305506 Plate 2 A09 CD95 DX2 Mouse IgG1, κ 305608 Plate 2 A10 CD96 NK92.39 Mouse IgG1, κ 338406 Plate 2 A11 CD97 VIM3b Mouse IgG1, κ 336308 Plate 2 A12 CD99 HCD99 Mouse IgG2a, κ 318008 Plate 2 B01 CD100 A8 Mouse IgG1, κ 328408 Plate 2 B02 CD101 (BB27) BB27 Mouse IgG1, κ 331006 Plate 2 B03 CD102 CBR-IC2/2 Mouse IgG2a, κ 328506 Plate 2 B04 CD103 Ber-ACT8 Mouse IgG1, κ 350206 Plate 2 B05 CD104 58XB4 Mouse IgG2a, κ 327808 Plate 2 B06 CD105 43A3 Mouse IgG1, κ 323206 Plate 2 B07 CD106 STA Mouse IgG1, κ 305806 Plate 2 B08 CD107a (LAMP-1) H4A3 Mouse IgG1, κ 328608 Plate 2 B09 CD108 MEM-150 Mouse IgM, κ 315704 Plate 2 B10 CD109 W7C5 Mouse IgG1, κ 323306 Plate 2 B11 CD111 R1.302 Mouse IgG1, κ 340404 Plate 2 B12 CD112 (Nectin-2) TX31 Mouse IgG1, κ 337410 Plate 2 C01 CD114 LMM741 Mouse IgG1, κ 346106 Plate 2 C02 CD115 9-4D2-1E4 Rat IgG1, κ 347304 Plate 2 C03 CD116 4H1 Mouse IgG1, κ 305908 Plate 2 C04 CD117 (c-kit) 104D2 Mouse IgG1, κ 313204 Plate 2 C05 CD119 (IFN-g Rd GIR-208 Mouse IgG1, κ 308606 chain) Plate 2 C06 CD122 TU27 Mouse IgG1, κ 339006 Plate 2 C07 CD123 6H6 Mouse IgG1, κ 306006 Plate 2 C08 CD124 G077F6 Mouse IgG2a, κ 355004 Plate 2 C09 CD126 (IL-6Rα) UV4 Mouse IgG1, κ 352804 Plate 2 C10 CD127 (IL-7Rα) A019D5 Mouse IgG1, κ 351304 Plate 2 C11 CD129 (IL-9 R) AH9R7 Mouse IgG2b, κ 310404 Plate 2 C12 CD131 1C1 Mouse IgG1, κ 306104 Plate 2 D01 CD132 TUGh4 Rat IgG2b, κ 338606 Plate 2 D02 CD134 Ber-ACT35 (ACT35) Mouse IgG1, κ 350004 Plate 2 D03 CD135 BV10A4H2 Mouse IgG1, κ 313306 Plate 2 D04 CD137 (4-1BB) 4B4-1 Mouse IgG1, κ 309804 Plate 2 D05 CD137L (4-1BB 5F4 Mouse IgG1, κ 311504 Ligand) Plate 2 D06 CD138 DL-101 Mouse IgG1, κ 352306 Plate 2 D07 CD140a 16A1 Mouse IgG1, κ 323506 Plate 2 D08 CD140b 18A2 Mouse IgG1, κ 323606 Plate 2 D09 CD141 M80 Mouse IgG1, κ 344104 Plate 2 D10 CD143 5-369 Mouse IgG1, κ 344204 Plate 2 D11 CD144 BV9 Mouse IgG2a, κ 348506 Plate 2 D12 CD146 SHM-57 Mouse IgG2a, κ 342004 Plate 2 E01 CD148 A3 Mouse IgG1, κ 328708 Plate 2 E02 CD150 (SLAM) A12 (7D4) Mouse IgG1, κ 306308 Plate 2 E03 CD152 L3D10 Mouse IgG1, κ 349906 Plate 2 E04 CD154 24-31 Mouse IgG1, κ 310806 Plate 2 E05 CD155 (PVR) SKII.4 Mouse IgG1, κ 337610 Plate 2 E06 CD156c (ADAM10) SHM14 Mouse IgG1, κ 352704 Plate 2 E07 CD158a/h HP-MA4 Mouse IgG2b, κ 339506 Plate 2 E08 CD158b (KIR2DL2/L3) DX27 Mouse IgG2a, κ 312606 Plate 2 E09 CD158d mAb 33 (33) Mouse IgG1, κ 347006 Plate 2 E10 CD158e1 (KIR3DL1) DX9 Mouse IgG1, κ 312708 Plate 2 E11 CD158f UP-R1 Mouse IgG1, κ 341304 Plate 2 E12 CD161 HP-3G10 Mouse IgG1, κ 339904 Plate 2 F01 CD162 KPL-1 Mouse IgG1, κ 328806 Plate 2 F02 CD163 GHI/61 Mouse IgG1, κ 333606 Plate 2 F03 CD164 67D2 Mouse IgG1, κ 324808 Plate 2 F04 CD165 SN2 (N6- D11) Mouse IgG1, κ 329010 Plate 2 F05 CD166 3A6 Mouse IgG1, κ 343904 Plate 2 F06 CD167a (DDR1) 51D6 Mouse IgG3, κ 334006 Plate 2 F07 CD169 7-239 Mouse IgG1, κ 346004 Plate 2 F08 CD170 (Siglec-5) 1A5 Mouse IgG1, κ 352004 Plate 2 F09 CD172a (SIRPa) SE5A5 Mouse IgG1, κ 323806 Plate 2 F10 CD172b (SIRPb) B4B6 Mouse IgG1, κ 323906 Plate 2 F11 CD172g (SIRPg) LSB2.20 Mouse IgG1, κ 336606 Plate 2 F12 CD178 (Fas-L) NOK-1 Mouse IgG1, κ 306407 Plate 2 G01 CD179a HSL96 Mouse IgG1, κ 347404 Plate 2 G02 CD179b HSL11 Mouse IgG1, κ 349804 Plate 2 G03 CD180 (RP105) MHR73-11 Mouse IgG1, κ 312906 Plate 2 G04 CD181 (CXCR1) 8F1/CXCR1 Mouse IgG2b, κ 320608 Plate 2 G05 CD182 (CXCR2) 5E8/CXCR2 Mouse IgG1, κ 320706 Plate 2 G06 CD183 G025H7 Mouse IgG1, κ 353706 Plate 2 G07 CD184 (CXCR4) 12G5 Mouse IgG2a, κ 306506 Plate 2 G08 CD193 (CCR3) 500000000 Mouse IgG2b, κ 310706 Plate 2 G09 CD195 (CCR5) T21/8 Mouse IgG1, κ 321406 Plate 2 G10 CD196 G034E3 Mouse IgG2b, κ 353410 Plate 2 G11 CD197 (CCR7) G043H7 Mouse IgG2a, κ 353204 Plate 2 G12 CD200 (OX2) OX-104 Mouse IgG1, κ 329206 Plate 2 H01 CD200 R OX-108 Mouse IgG1, κ 329306 Plate 2 H02 CD201 (EPCR) RCR-401 Rat IgG1, κ 351904 Plate 2 H03 CD202b (Tie2/Tek) 33.1 (Ab33) Mouse IgG1, κ 334206 Plate 2 H04 CD203c (E-NPP3) NP4D6 Mouse IgG1, κ 324606 Plate 2 H05 CD205 (DEC- 205) HD30 Mouse IgG1, κ 342204 Plate 2 H06 CD206 (MMR) 15-2 Mouse IgG1, κ 321106 Plate 2 H07 CD207 (Langerin) 10E2 Mouse IgG1, κ 352204 Plate 2 H08 CD209 (DC- SIGN) 9E9A8 Mouse IgG2a, κ 330106 Plate 2 H09 CD210 (IL- 10 R) 3F9 Rat IgG2a, κ 308804 Plate 2 H10 CD213a2 SHM38 Mouse IgG1, κ 354404 Plate 2 H11 CD215 (IL- 15Rα) JM7A4 Mouse IgG2b, κ 330208 Plate 2 H12 CD218a (IL-18Rα) H44 Mouse IgG1, κ 313808 Plate 3 A01 Blank Plate 3 A02 CD220 B6.220 Mouse IgG2b,.sub.— 352604 Plate 3 A03 CD221 (IGF-1R) 1H7/CD221 Mouse IgG1,.sub.— 351806 Plate 3 A04 CD226 (DNAM-1) 11A8 Mouse IgG1,.sub.— 338306 Plate 3 A05 CD229 (Ly-9) HLy-9.1.25 Mouse IgG1,.sub.— 326108 Plate 3 A06 CD231 (TALLA) SN1a (M3-3D9) Mouse IgG1,.sub.— 329406 Plate 3 A07 CD235ab HIR2 Mouse IgG2b,.sub.— 306604 Plate 3 A08 CD243 UIC2 Mouse IgG2a,.sub.— 348606 Plate 3 A09 CD244 (2B4) C1.7 Mouse IgG1,.sub.— 329508 Plate 3 A10 CD245 (p220/240) DY12 Mouse IgG1,.sub.— Inquire Plate 3 A11 CD252 (OX40L) 11C3.1 Mouse IgG1,.sub.— 326308 Plate 3 A12 CD253 (Trail) RIK-2 Mouse IgG1,.sub.— 308206 Plate 3 B01 CD254 MIH24 Mouse IgG1,.sub.— 347504 Plate 3 B02 CD255 (TWEAK) CARL-1 Mouse IgG3,.sub.— 308305 Plate 3 B03 CD257 (BAFF, BLYS) T7-241 Mouse IgG1,.sub.— 318606 Plate 3 B04 CD258 (LIGHT) T5-39 Mouse IgG2a,.sub.— 318706 Plate 3 B05 CD261 (DR4, TRAIL-R1) DJR1 Mouse IgG1,.sub.— 307206 Plate 3 B06 CD262 (DR5, TRAIL-R2) DJR2-4 (7-8) Mouse IgG1,.sub.— 307406 Plate 3 B07 CD263 (DcR1, TRAIL-R3) DJR3 Mouse IgG1,.sub.— 307006 Plate 3 B08 CD266 (Fn14) ITEM-1 Mouse IgG1,.sub.— 314004 Plate 3 B09 CD267 (TACI) 1A1 Rat IgG2a,.sub.— 311906 Plate 3 B10 CD268 (BAFF-R, BAFFR) 11C1 Mouse IgG1,.sub.— 316906 Plate 3 B11 CD270 (HVEM) 122 Mouse IgG1,.sub.— 318806 Plate 3 B12 CD271 ME20.4 Mouse IgG1,.sub.— 345106 Plate 3 C01 CD273 (B7- DC, PD-L2) 24F.10C12 Mouse IgG2a,.sub.— 329606 Plate 3 C02 CD274 (B7- H1, PD-L1) 29E.2A3 Mouse IgG2b,.sub.— 329706 Plate 3 C03 CD275 (B7- H2, B7-RP1) 9F.8A4 Mouse IgG1,.sub.— 329806 Plate 3 C04 CD276 MIH42 Mouse IgG1,.sub.— 351004 Plate 3 C05 CD277 BT3.1 Mouse IgG1,.sub.— 342704 Plate 3 C06 CD278 (ICOS) C398.4A Arm. Hamster IgG 313508 Plate 3 C07 CD279 (PD-1) EH12.2H7 Mouse IgG1,.sub.— 329906 Plate 3 C08 CD282 (TLR2) TL2.1 Mouse IgG2a,.sub.— 309708 Plate 3 C09 CD284 (TLR4) HTA125 Mouse IgG2a,.sub.— 312806 Plate 3 C10 CD286 (TLR6) TLR6.127 Mouse IgG1,.sub.— 334708 Plate 3 C11 CD290 3C10C5 Mouse IgG1,.sub.— 354604 Plate 3 C12 CD294 BM16 Rat IgG2a,.sub.— 350106 Plate 3 D01 CD298 LNH-94 Mouse IgG1,.sub.— 341704 Plate 3 D02 CD300e (IREM-2) UP-H2 Mouse IgG1,.sub.— 339704 Plate 3 D03 CD300F UP-D2 Mouse IgG1,.sub.— 340604 Plate 3 D04 CD301 H037G3 Mouse IgG2a,.sub.— 354704 Plate 3 D05 CD303 201A Mouse IgG2a,.sub.— 354204 Plate 3 D06 CD304 12C2 Mouse IgG2a,.sub.— 354504 Plate 3 D07 CD307 509f6 Mouse IgG2a,.sub.— 340304 Plate 3 D08 CD307d (FcRL4) 413D12 Mouse IgG2b,.sub.— 340204 Plate 3 D09 CD314 (NKG2D) 1D11 Mouse IgG1,.sub.— 320806 Plate 3 D10 CD317 RS38E Mouse IgG1,.sub.— 348406 Plate 3 D11 CD318 (CDCP1) CUB1 Mouse IgG2b,.sub.— 324006 Plate 3 D12 CD319 (CRACC) 162.1 Mouse IgG2b,.sub.— 331806 Plate 3 E01 CD324 (E-Cadherin) 67A4 Mouse IgG1,.sub.— 324106 Plate 3 E02 CD325 8C11 Mouse IgG1,.sub.— 350805 Plate 3 E03 CD326 (Ep- CAM) 9C4 Mouse IgG2b,.sub.— 324206 Plate 3 E04 CD328 (Siglec-7) 6-434 Mouse IgG1,.sub.— 339204 Plate 3 E05 CD334 (FGFR4) 4FR6D3 Mouse IgG1,.sub.— 324306 Plate 3 E06 CD335 (NKp46) 9.00E+02 Mouse IgG1,.sub.— 331908 Plate 3 E07 CD336 (NKp44) P44-8 Mouse IgG1,.sub.— 325108 Plate 3 E08 CD337 (NKp30) P30-15 Mouse IgG1,.sub.— 325208 Plate 3 E09 CD338 (ABCG2) 5D3 Mouse IgG2b,.sub.— 332008 Plate 3 E10 CD340 (erbB2/HER-2) 24D2 Mouse IgG1,.sub.— 324406 Plate 3 E11 CD344 (Frizzled-4) CH3A4A7 Mouse IgG1,.sub.— 326606 Plate 3 E12 CD351 TX61 Mouse IgG1,.sub.— 137306 Plate 3 F01 CD352 (NTB-A) NT-7 Mouse IgG1,.sub.— 317208 Plate 3 F02 CD354 (TREM-1) TREM-26 Mouse IgG1,.sub.— 314906 Plate 3 F03 CD355 (CRTAM) Cr24.1 Mouse IgG2a,.sub.— 339106 Plate 3 F04 CD357 (GITR) 621 Mouse IgG1,.sub.— 311604 Plate 3 F05 CD360 (IL- 21R) 2G1-K12 Mouse IgG1,.sub.— 347806 Plate 3 F06 _2- micro- globulin 2M2 Mouse IgG1,.sub.— 316306 Plate 3 F07 BTLA MIH26 Mouse IgG2a,.sub.— 344506 Plate 3 F08 C3AR hC3aRZ8 Mouse IgG2b 345804 Plate 3 F09 C5L2 1D9-M12 Mouse IgG2a,.sub.— 342404 Plate 3 F10 CCR10 May-88 Arm. hamster IgG 341504 Plate 3 F11 CLEC12A 50C1 Mouse IgG2a,.sub.— 353604 Plate 3 F12 CLEC9A 8F9 Mouse IgG2a,.sub.— 353804 Plate 3 G01 CX3CR1 2A9-1 Rat IgG2b,.sub.— 341604 Plate 3 G02 CXCR7 8F11-M16 Mouse IgG2b,.sub.— 331104 Plate 3 G03 _-Opioid Receptor DOR7D2A4 Mouse IgG2b,.sub.— 327206 Plate 3 G04 DLL1 MHD1-314 Mouse IgG1,.sub.— 346404 Plate 3 G05 DLL4 MHD4-46 Mouse IgG1,.sub.— 346506 Plate 3 G06 DR3 (TRAMP) JD3 Mouse IgG1,.sub.— 307106 Plate 3 G07 EGFR AY13 Mouse IgG1,.sub.— 352904 Plate 3 G08 erbB3/HER-3 1B4C3 Mouse IgG2a,.sub.— 324706 Plate 3 G09 Fc_Rl.sub.— AER-37 (CRA-1) Mouse IgG2b,.sub.— 334610 Plate 3 G10 FcRL6 2H3 Mouse IgG2b,.sub.— Inquire Plate 3 G11 Galectin-9 9M1-3 Mouse IgG1,.sub.— 348906 Plate 3 G12 GARP (LRRC32) 7B11 Mouse IgG2b,.sub.— 352504 Plate 3 H01 HLA-A, B, C W6/32 Mouse IgG2a,.sub.— 311406 Plate 3 H02 HLA-A2 BB7.2 Mouse IgG2b,.sub.— 343306 Plate 3 H03 HLA-DQ HLADQ1 Mouse IgG1,.sub.— 318106 Plate 3 H04 HLA-DR L243 Mouse IgG2a,.sub.— 307606 Plate 3 H05 HLA-E 3D12 Mouse IgG1,.sub.— 342604 Plate 3 H06 HLA-G 87G Mouse IgG2a,.sub.— 335906 Plate 3 H07 IFN-g R b chain 2HUB-159 Hamster IgG 308504 Plate 3 H08 Ig light chain k MHK-49 Mouse IgG1,.sub.— 316508 Plate 3 H09 Ig light chain.sub.— MHL-38 Mouse IgG2a,.sub.— 316608 Plate 3 H10 IgD IA6-2 Mouse IgG2a,.sub.— 348204 Plate 3 H11 IgM MHM-88 Mouse IgG1,.sub.— 314508 Plate 3 H12 IL-28RA MHLICR2a Mouse IgG2a,.sub.— 337804 Plate 4 A01 Blank Plate 4 A02 Integrin α9β1 Y9A2 Mouse IgG1, κ 351606 Plate 4 A03 integrin β5 AST-3T Mouse IgG2a, κ 345204 Plate 4 A04 integrin β7 FIB504 Rat IgG2a, κ 321204 Plate 4 A05 Jagged 2 MHJ2-523 Mouse IgG1, κ 346904 Plate 4 A06 LAP TW4-6H10 Mouse IgG1, κ 349704 Plate 4 A07 Lymphotoxin b Receptor 31G4D8 Mouse IgG2b, κ 322008 Plate 4 A08 Mac-2 (Ga- lectin-3) Gal397 Mouse IgG1, κ 126705 Plate 4 A09 MAIR-II TX45 Mouse IgG1, κ 334804 Plate 4 A10 MICA/MICB 6D4 Mouse IgG2a, κ 320906 Plate 4 A11 MSC (W3D5) W3D5 Mouse IgG2a, κ 327506 Plate 4 A12 MSC (W5C5) W5C5 Mouse IgG1, κ 327406 Plate 4 B01 MSC (W7C6) W7C6 Mouse IgG1, κ 327606 Plate 4 B02 MSC and NPC (W4A5) W4A5 Mouse IgG1, κ 330806 Plate 4 B03 MSCA-1 (MSC, W8B2) W8B2 Mouse IgG1, κ 327306 Plate 4 B04 NKp80 5D12 Mouse IgG1, κ 346706 Plate 4 B05 Notch 1 MHN1-519 Mouse IgG1, κ 352106 Plate 4 B06 Notch 2 MHN2-25 Mouse IgG2a, κ 348304 Plate 4 B07 Notch 3 MHN3-21 Mouse IgG1, κ 345406 Plate 4 B08 Notch 4 MHN4-2 Mouse IgG1, κ 349004 Plate 4 B09 NPC (57D2) 57D2 Mouse IgG1, κ 327706 Plate 4 B10 Podoplanin NC-08 Rat IgG2a, λ 337004 Plate 4 B11 Pre-BCR HSL2 Mouse IgG1, κ 347904 Plate 4 B12 PSMA LNI-17 Mouse IgG1, κ 342504 Plate 4 C01 Siglec-10 5G6 Mouse IgG1, κ 347604 Plate 4 C02 Siglec-8 7C9 Mouse IgG1, κ 347104 Plate 4 C03 Siglec-9 K8 Mouse IgG1, κ 351504 Plate 4 C04 SSEA-1 MC-480 Mouse IgM, κ 125606 Plate 4 C05 SSEA-3 MC-631 Rat IgM, κ 330312 Plate 4 C06 SSEA-4 MC-813-70 Mouse IgG3, κ 330406 Plate 4 C07 SSEA-5 8E+11 Mouse IgG1, κ 355204 Plate 4 C08 TCR g/d B1 Mouse IgG1, κ 331210 Plate 4 C09 TCR Vβ13.2 H132 Mouse IgG1, κ 333108 Plate 4 C10 TCR Vβ23 αHUT7 Mouse IgG1, κ 349406 Plate 4 C11 TCR Vβ8 JR2 (JR.2) Mouse IgG2b, κ 348104 Plate 4 C12 TCR Vβ9 MKB1 Mouse IgG2b, κ 349204 Plate 4 D01 TCR Vδ2 B6 Mouse IgG1, κ 331408 Plate 4 D02 TCR Vg9 B3 Mouse IgG1, κ 331308 Plate 4 D03 TCR Vα24- Jα18 6B11 Mouse IgG1, κ 342904 Plate 4 D04 TCR Vα7.2 3C10 Mouse IgG1, κ 351706 Plate 4 D05 TCR α/β IP26 Mouse IgG1, κ 306708 Plate 4 D06 Tim-1 1D12 Mouse IgG1, κ 353904 Plate 4 D07 Tim-3 F38-2E2 Mouse IgG1, κ 345006 Plate 4 D08 Tim-4 9F4 Mouse IgG1, κ 354004 Plate 4 D09 TLT-2 MIH61 Mouse IgG1, κ 351104 Plate 4 D10 TRA-1-60-R TRA-1-60-R Mouse IgM, κ 330610 Plate 4 D11 TRA-1-81 TRA-1-81 Mouse IgM, κ 330708 Plate 4 D12 TSLPR (TSLP-R) 1B4 Mouse IgG1, κ 322806 Plate 4 E01 Ms IgG1, κ ITCL MOPC-21 Mouse IgG1, κ 400112 Plate 4 E02 Ms IgG2a, κ ITCL MOPC-173 Mouse IgG2a, κ 400212 Plate 4 E03 Ms IgG2b, κ ITCL MPC-11 Mouse IgG2b, κ 400314 Plate 4 E04 Ms IgG3, κ ITCL MG3-35 Mouse IgG3, κ 401320 Plate 4 E05 Ms IgM, κ ITCL MM-30 Mouse IgM, κ 401609 Plate 4 E06 Rat IgG1, κ ITCL RTK2071 Rat IgG1, κ 400408 Plate 4 E07 Rat IgG2a, κ ITCL RTK2758 Rat IgG2a, κ 400508 Plate 4 E08 Rat IgG2b, κ ITCL RTK4530 Rat IgG2b, κ 400636 Plate 4 E09 Rat IgM, κ ITCL RTK2118 Rat IgM, κ 400808 Plate 4 E10 AH IgG, ITCL HTK888 Arm. Hamster IgG 400907 Plate 4 E11 CD177 MEM-166 mouse IgG1, k 315806 Plate 4 E12 IL1RAP 89412 mouse IgG1, k FAB676P Screen Iteration 70007 Plate WELL ID Specificity Clone Isotype Plate 1 A01 Blank Plate 1 A02 IgG Isotype Ctrl HTK888 Arm. Hamster IgG Plate 1 A03 CCR10 6588-5 Arm. Hamster IgG Plate 1 A04 CD278 C398.4A Arm. Hamster IgG Plate 1 A05 IFN-γ R b chain 2HUB-159 Hamster IgG Plate 1 A06 IgG1, κ Isotype Ctrl MOPC-21 Mouse IgG1, k Plate 1 A07 CD46 TRA-2-10 Mouse IgG1 Plate 1 A08 CD70 113-16 Mouse IgG1 Plate 1 A09 CD1a HI149 mouse IgG1, k Plate 1 A10 CD2 RPA-2.10 mouse IgG1, k Plate 1 A11 β2-microglobulin 2M2 mouse IgG1, k Plate 1 A12 B7-H4 MIH43 mouse IgG1, k Plate 1 B01 Cadherin 11 16G5 mouse IgG1, k Plate 1 B02 CD10 HI10a mouse IgG1, k Plate 1 B03 CD100 A8 mouse IgG1, k Plate 1 B04 CD103 Ber-ACT8 mouse IgG1, k Plate 1 B05 CD105 (Endoglin) SN6h mouse IgG1, k Plate 1 B06 CD106 STA mouse IgG1, k Plate 1 B07 CD107a H4A3 mouse IgG1, k Plate 1 B08 CD107b H4B4 mouse IgG1, k Plate 1 B09 CD109 W7C5 mouse IgG1, k Plate 1 B10 CD111 R1.302 mouse IgG1, k Plate 1 B11 CD112 TX31 mouse IgG1, k Plate 1 B12 CD114 LMM741 mouse IgG1, k Plate 1 C01 CD116 4H1 mouse IgG1, k Plate 1 C02 CD117 104D2 mouse IgG1, k Plate 1 C03 CD119 GIR-208 mouse IgG1, k Plate 1 C04 CD11a HI111 mouse IgG1, k Plate 1 C05 CD11b ICRF44 mouse IgG1, k Plate 1 C06 CD122 TU27 mouse IgG1, k Plate 1 C07 CD123 6H6 mouse IgG1, k Plate 1 C08 CD126 UV4 mouse IgG1, k Plate 1 C09 CD127 A019D5 mouse IgG1, k Plate 1 C10 CD13 WM15 mouse IgG1, k Plate 1 C11 CD131 1C1 mouse IgG1, k Plate 1 C12 CD134 Ber-ACT35 (ACT35) mouse IgG1, k Plate 1 D01 CD135 BV10A4H2 mouse IgG1, k Plate 1 D02 CD137 4B4-1 mouse IgG1, k Plate 1 D03 4-1BB Ligand 5F4 mouse IgG1, k Plate 1 D04 CD138 MI15 mouse IgG1, k Plate 1 D05 CD14 63D3 mouse IgG1, k Plate 1 D06 CD140a 16A1 mouse IgG1, k Plate 1 D07 CD140b 18A2 mouse IgG1, k Plate 1 D08 CD141 M80 mouse IgG1, k Plate 1 D09 CD142 NY2 mouse IgG1, k Plate 1 D10 CD143 5-369 mouse IgG1, k Plate 1 D11 CD146 P1H12 mouse IgG1, k Plate 1 D12 CD148 A3 mouse IgG1, k Plate 1 E01 CD15 W6D3 mouse IgG1, k Plate 1 E02 CD150 A12 (7D4) mouse IgG1, k Plate 1 E03 CD151 50-6 mouse IgG1, k Plate 1 E04 CD154 24-31 mouse IgG1, k Plate 1 E05 CD156c SHM14 mouse IgG1, k Plate 1 E06 CD158e1 DX9 mouse IgG1, k Plate 1 E07 CD16 3G8 mouse IgG1, k Plate 1 E08 CD161 HP-3G10 mouse IgG1, k Plate 1 E09 CD162 KPL-1 mouse IgG1, k Plate 1 E10 CD163 GHI/61 mouse IgG1, k Plate 1 E11 CD164 67D2 mouse IgG1, k Plate 1 E12 CD165 SN2 (N6- D11) mouse IgG1, k Plate 1 F01 CD166 3A6 mouse IgG1, k Plate 1 F02 CD169 7-239 mouse IgG1, k Plate 1 F03 CD170 1A5 mouse IgG1, k Plate 1 F04 CD172a/b (SIRPα/β) SE5A5 mouse IgG1, k Plate 1 F05 CD172g (SIRPy) LSB2.20 mouse IgG1, k Plate 1 F06 CD178 NOK-1 mouse IgG1, k Plate 1 F07 CD179a HSL96 mouse IgG1, k Plate 1 F08 CD179b HSL11 mouse IgG1, k Plate 1 F09 CD18 TS1/18 mouse IgG1, k Plate 1 F10 CD180 MHR73-11 mouse IgG1, k Plate 1 F11 CD182 5E8/CXCR2 mouse IgG1, k Plate 1 F12 CD183 G025H7 mouse IgG1, k Plate 1 G01 CD185 J252D4 mouse IgG1, k Plate 1 G02 CD19 HIB19 mouse IgG1, k Plate 1 G03 CD191 5F10B29 mouse IgG1, k Plate 1 G04 CD194 L291H4 mouse IgG1, k Plate 1 G05 CD1b SN13 (K5- 1B8) mouse IgG1, k Plate 1 G06 CD1c L161 mouse IgG1, k Plate 1 G07 CD200 OX-104 mouse IgG1, k Plate 1 G08 CD200R OX-108 mouse IgG1, k Plate 1 G09 CD202b 33.1 (Ab33) mouse IgG1, k Plate 1 G10 CD203c NP4D6 mouse IgG1, k Plate 1 G11 CD205 HD83 mouse IgG1, k Plate 1 G12 CD206 15-2 mouse IgG1, k Plate 1 H01 CD207 1E3 mouse IgG1, k Plate 1 H02 CD21 Bu32 mouse IgG1, k Plate 1 H03 CD213a1 SS12B mouse IgG1, k Plate 1 H04 CD213a2 SHM38 mouse IgG1, k Plate 1 H05 CD218a H44 mouse IgG1, k Plate 1 H06 CD221 1H7/CD221 mouse IgG1, k Plate 1 H07 CD223 (LAG-3) 11C3C65 mouse IgG1, k Plate 1 H08 CD226 11A8 mouse IgG1, k Plate 1 H09 CD227 16A mouse IgG1, k Plate 1 H10 CD229 HLy-9.1.25 mouse IgG1, k Plate 1 H11 CD23 EBVCS-5 mouse IgG1, k Plate 1 H12 CD231 SN1a (M3- 3D9) mouse IgG1, k Plate 2 A01 Blank Plate 2 A02 CD244 (2B4) C1.7 mouse IgG1, k Plate 2 A03 CD245 DY12 mouse IgG1, k Plate 2 A04 CD25 M-A251 mouse IgG1, k Plate 2 A05 CD252 11C3.1 mouse IgG1, k Plate 2 A06 CD261 DJR1 mouse IgG1, k Plate 2 A07 CD262 DJR2-4 (7-8) mouse IgG1, k Plate 2 A08 CD263 DJR3 mouse IgG1, k Plate 2 A09 CD266 ITEM-1 mouse IgG1, k Plate 2 A10 CD268 11C1 mouse IgG1, k Plate 2 A11 CD27 M-T271 mouse IgG1, k Plate 2 A12 CD271 ME20.4 mouse IgG1, k Plate 2 B01 CD275 9F.8A4 mouse IgG1, k Plate 2 B02 CD276 MIH42 mouse IgG1, k Plate 2 B03 CD277 BT3.1 mouse IgG1, k Plate 2 B04 CD279 EH12.2H7 mouse IgG1, k Plate 2 B05 CD28 CD28.2 mouse IgG1, k Plate 2 B06 CD29 TS2/16 mouse IgG1, k Plate 2 B07 CD290 3C10C5 mouse IgG1, k Plate 2 B08 CD298 LNH-94 mouse IgG1, k Plate 2 B09 CD3 UCHT1 mouse IgG1, k Plate 2 B10 CD30 BY88 mouse IgG1, k Plate 2 B11 CD300c TX45 mouse IgG1, k Plate 2 B12 CD309 7D4-6 mouse IgG1, k Plate 2 C01 CD31 WM59 mouse IgG1, k Plate 2 C02 CD314 1D11 mouse IgG1, k Plate 2 C03 CD317 RS38E mouse IgG1, k Plate 2 C04 CD324 67A4 mouse IgG1, k Plate 2 C05 CD325 8C11 mouse IgG1, k Plate 2 C06 CD328 6-434 mouse IgG1, k Plate 2 C07 CD33 WM53 mouse IgG1, k Plate 2 C08 CD334 4FR6D3 mouse IgG1, k Plate 2 C09 CD335 9E2 mouse IgG1, k Plate 2 C10 CD336 P44-8 mouse IgG1, k Plate 2 C11 CD337 P30-15 mouse IgG1, k Plate 2 C12 CD34 581 mouse IgG1, k Plate 2 D01 CD340 24D2 mouse IgG1, k Plate 2 D02 CD344 CH3A4A7 mouse IgG1, k Plate 2 D03 CD35 E11 mouse IgG1, k Plate 2 D04 CD354 TREM-26 mouse IgG1, k Plate 2 D05 CD360 17A12 mouse IgG1, k Plate 2 D06 CD365 1D12 mouse IgG1, k Plate 2 D07 CD366 F38-2E2 mouse IgG1, k Plate 2 D08 CLEC4A 9E8 mouse IgG1, k Plate 2 D09 CD36L1 m1b9 mouse IgG1, k Plate 2 D10 CD38 HIT2 mouse IgG1, k Plate 2 D11 CD39 A1 mouse IgG1, k Plate 2 D12 CD4 RPA-T4 mouse IgG1, k Plate 2 E01 CD40 5C3 mouse IgG1, k Plate 2 E02 CD41 HIP8 mouse IgG1, k Plate 2 E03 CD42b HIP1 mouse IgG1, k Plate 2 E04 CD43 CD43-10G7 mouse IgG1, k Plate 2 E05 CD44 BJ18 mouse IgG1, k Plate 2 E06 CD45 HI30 mouse IgG1, k Plate 2 E07 CD47 CC2C6 mouse IgG1, k Plate 2 E08 CD48 BJ40 mouse IgG1, k Plate 2 E09 CD49a TS2/7 mouse IgG1, k Plate 2 E10 CD49b P1E6-C5 mouse IgG1, k Plate 2 E11 CD49c ASC-1 mouse IgG1, k Plate 2 E12 CD49d 9F10 mouse IgG1, k Plate 2 F01 CD5 UCHT2 mouse IgG1, k Plate 2 F02 CD50 CBR-IC3/1 mouse IgG1, k Plate 2 F03 CD54 HA58 mouse IgG1, k Plate 2 F04 CD55 JS11 mouse IgG1, k Plate 2 F05 CD56 (NCAM) 5.1H11 mouse IgG1, k Plate 2 F06 CD58 TS2/9 mouse IgG1, k Plate 2 F07 CD6 BL-CD6 mouse IgG1, k Plate 2 F08 CD61 VI-PL2 mouse IgG1, k Plate 2 F09 CD62E HAE-1f mouse IgG1, k Plate 2 F10 CD62L DREG-56 mouse IgG1, k Plate 2 F11 CD62P AK4 mouse IgG1, k Plate 2 F12 CD63 H5C6 mouse IgG1, k Plate 2 G01 CD64 10.1 mouse IgG1, k Plate 2 G02 CD69 FN50 mouse IgG1, k Plate 2 G03 CD73 AD2 mouse IgG1, k Plate 2 G04 CD74 LN2 mouse IgG1, k Plate 2 G05 CD79b CB3-1 mouse IgG1, k Plate 2 G06 CD8a SK1 mouse IgG1, k Plate 2 G07 CD80 2D10 mouse IgG1, k Plate 2 G08 CD81 5A6 mouse IgG1, k Plate 2 G09 CD82 ASL-24 mouse IgG1, k Plate 2 G10 CD83 HB15e mouse IgG1, k Plate 2 G11 CD85 17G10.2 mouse IgG1, k Plate 2 G12 CD85k ZM4.1 mouse IgG1, k Plate 2 H01 CD87 VIM5 mouse IgG1, k Plate 2 H02 CD89 A59 mouse IgG1, k Plate 2 H03 CD8a RPA-T8 mouse IgG1, k Plate 2 H04 CD9 HI9a mouse IgG1, k Plate 2 H05 CD90 5E10 mouse IgG1, k Plate 2 H06 CD93 VIMD2 mouse IgG1, k Plate 2 H07 CD94 DX22 mouse IgG1, k Plate 2 H08 CD95 DX2 mouse IgG1, k Plate 2 H09 CD96 NK92.39 mouse IgG1, k Plate 2 H10 CD97 VIM3b mouse IgG1, k Plate 2 H11 CD99 3B2/TA8 mouse IgG1, k Plate 2 H12 CXCL16 22-19-12 mouse IgG1, k Plate 3 A01 Blank Plate 3 A02 DLL1 MHD1-314 mouse IgG1, k Plate 3 A03 DLL4 MHD4-46 mouse IgG1, k Plate 3 A04 DR3 JD3 mouse IgG1, k Plate 3 A05 EGFR AY13 mouse IgG1, k Plate 3 A06 CD357 108-17 mouse IgG1, k Plate 3 A07 GPR19 K152D10 mouse IgG1, k Plate 3 A08 GPR56 CG4 mouse IgG1, k Plate 3 A09 HLA-E 3D12 mouse IgG1, k Plate 3 A10 HVEM 122 mouse IgG1, k Plate 3 A11 Ig light chain κ MHK-49 mouse IgG1, k Plate 3 A12 IgM MHM-88 mouse IgG1, k Plate 3 B01 CD360 2G1-K12 mouse IgG1, k Plate 3 B02 Integrin α9β1 Y9A2 mouse IgG1, k Plate 3 B03 Jagged 2 MHJ2-523 mouse IgG1, k Plate 3 B04 Ksp37 TDA3 mouse IgG1, k Plate 3 B05 LAP TW4-2F8 mouse IgG1, k Plate 3 B06 LY6G6D 13.8 mouse IgG1, k Plate 3 B07 MERTK 590H11G1E3 mouse IgG1, k Plate 3 B08 MSC W7C6 mouse IgG1, k Plate 3 B09 MSC, NPC W4A5 mouse IgG1, k Plate 3 B10 TNAP W8B2 mouse IgG1, k Plate 3 B11 MUC-13 TCC16 mouse IgG1, k Plate 3 B12 NKp80 5D12 mouse IgG1, k Plate 3 C01 Notch 1 MHN1-519 mouse IgG1, k Plate 3 C02 Notch3 MHN3-21 mouse IgG1, k Plate 3 C03 Notch 4 MHN4-2 mouse IgG1, k Plate 3 C04 NPC 57D2 mouse IgG1, k Plate 3 C05 CD352 NT-7 mouse IgG1, k Plate 3 C06 PSMA LNI-17 mouse IgG1, k Plate 3 C07 ROR1 2A2 mouse IgG1, k Plate 3 C08 Siglec-10 5G6 mouse IgG1, k Plate 3 C09 CD328 S7.7 mouse IgG1, k Plate 3 C10 Siglec-8 7C9 mouse IgG1, k Plate 3 C11 Siglec-9 K8 mouse IgG1, k Plate 3 C12 SSEA-5 8E11 mouse IgG1, k Plate 3 D01 SUSD2 W5C5 mouse IgG1, k Plate 3 D02 TCR α/β IP26 mouse IgG1, k Plate 3 D03 TCR γ/δ B1 mouse IgG1, k Plate 3 D04 Tim-4 9F4 mouse IgG1, k Plate 3 D05 TLT-2 MIH61 mouse IgG1, k Plate 3 D06 TM4SF20 C9 mouse IgG1, k Plate 3 D07 TRA-2-49 TRA-2- 49/6E mouse IgG1, k Plate 3 D08 TRA-2-54 TRA-2-54/2J mouse IgG1, k Plate 3 D09 TSLPR 1B4 mouse IgG1, k Plate 3 D10 VEGFR-3 9D9F9 mouse IgG1, k Plate 3 D11 IgG2a, κ Isotype Ctrl MOPC-173 mouse IgG2a, k Plate 3 D12 APCDD1 7.13 mouse IgG2a, k Plate 3 E01 CD272 MIH26 mouse IgG2a, k Plate 3 E02 CD198 L263G8 mouse IgG2a, k Plate 3 E03 CCRL2 K097F7 mouse IgG2a, k Plate 3 E04 CD102 CBR-IC2/2 mouse IgG2a, k Plate 3 E05 CD104 58XB4 mouse IgG2a, k Plate 3 E06 CD124 G077F6 mouse IgG2a, k Plate 3 E07 CD130 2E1B02 mouse IgG2a, k Plate 3 E08 CD144 BV9 mouse IgG2a, k Plate 3 E09 CD152 (CTLA-4) BNI3 mouse IgG2a, k Plate 3 E10 CD155 TX24 mouse IgG2a, k Plate 3 E11 CD158b DX27 mouse IgG2a, k Plate 3 E12 CD184 12G5 mouse IgG2a, k Plate 3 F01 CD186 K041E5 mouse IgG2a, k Plate 3 F02 CD192 K036C2 mouse IgG2a, k Plate 3 F03 CD197 G043H7 mouse IgG2a, k Plate 3 F04 CD199 L053E8 mouse IgG2a, k Plate 3 F05 CD209 9E9A8 mouse IgG2a, k Plate 3 F06 CD217 W15177A mouse IgG2a, k Plate 3 F07 CD230 (Prion) 3F4 mouse IgG2a, k Plate 3 F08 CD24 ML5 mouse IgG2a, k Plate 3 F09 CD243 UIC2 mouse IgG2a, k Plate 3 F10 CD26 BA5b mouse IgG2a, k Plate 3 F11 CD269 19F2 mouse IgG2a, k Plate 3 F12 CD282 TL2.1 mouse IgG2a, k Plate 3 G01 CD284 HTA125 mouse IgG2a, k Plate 3 G02 CD301 H037G3 mouse IgG2a, k Plate 3 G03 CD303 201A mouse IgG2a, k Plate 3 G04 CD304 12C2 mouse IgG2a, k Plate 3 G05 CD307e 509f6 mouse IgG2a, k Plate 3 G06 CD323 SHM33 mouse IgG2a, k Plate 3 G07 CD357 108-17 mouse IgG2a, k Plate 3 G08 CD36 5-271 mouse IgG2a, k Plate 3 G09 CD369 15E2 mouse IgG2a, k Plate 3 G10 CD370 8F9 mouse IgG2a, k Plate 3 G11 CD371 50C1 mouse IgG2a, k Plate 3 G12 CD45RO UCHL1 mouse IgG2a, k Plate 3 H01 CD51 NKI-M9 mouse IgG2a, k Plate 3 H02 CD59 p282 (H19) mouse IgG2a, k Plate 3 H03 CD7 CD7-6B7 mouse IgG2a, k Plate 3 H04 CD71 CY1G4 mouse IgG2a, k Plate 3 H05 CD84 CD84.1.21 mouse IgG2a, k Plate 3 H06 CD88 S5/1 mouse IgG2a, k Plate 3 H07 CD355 Cr24.1 mouse IgG2a, k Plate 3 H08 erbB3 1B4C3 mouse IgG2a, k Plate 3 H09 FPR3 K102B9 mouse IgG2a, k Plate 3 H10 Ganglioside GD2 14G2a mouse IgG2a, k Plate 3 H11 GPR83 K07JP05 mouse IgG2a, k Plate 3 H12 HLA-A, B, C W6/32 mouse IgG2a, k Plate 4 A01 Blank Plate 4 A02 HLA-DR L243 mouse IgG2a, k Plate 4 A03 Ig light chain λ MHL-38 mouse IgG2a, k Plate 4 A04 IgD IA6-2 mouse IgG2a, k Plate 4 A05 IL-28RA MHLICR2a mouse IgG2a, k Plate 4 A06 integrin β5 AST-3T mouse IgG2a, k Plate 4 A07 KLRG1 SA231A2 mouse IgG2a, k Plate 4 A08 LOX-1 15C4 mouse IgG2a, k Plate 4 A09 MICA/MICB 6D4 mouse IgG2a, k Plate 4 A10 SUSD2 W3D5 mouse IgG2a, k Plate 4 A11 Notch 2 MHN2-25 mouse IgG2a, k Plate 4 A12 TACSTD2 NY18 mouse IgG2a, k Plate 4 B01 TIGIT (VSTM3) A15153G mouse IgG2a, k Plate 4 B02 IgG2b, κ Isotype Ctrl MPC-11 mouse IgG2b, k Plate 4 B03 C3aR hC3aRZ8 mouse IgG2b, k Plate 4 B04 CCX-CKR (CCRL1) 13E11 mouse IgG2b, k Plate 4 B05 CD110 S-HCL-3 mouse IgG2b, k Plate 4 B06 CD129 AH9R7 mouse IgG2b, k Plate 4 B07 CD158 HP-MA4 mouse IgG2b, k Plate 4 B08 CD181 8F1/CXCR1 mouse IgG2b, k Plate 4 B09 CD193 5E8 mouse IgG2b, k Plate 4 B10 CD196 G034E3 mouse IgG2b, k Plate 4 B11 CD1d 51.1 mouse IgG2b, k Plate 4 B12 CD20 2H7 mouse IgG2b, k Plate 4 C01 CD22 S-HCL-1 mouse IgG2b, k Plate 4 C02 CD220 B6.220 mouse IgG2b, k Plate 4 C03 CD235ab HIR2 mouse IgG2b, k Plate 4 C04 CD258 T5-39 mouse IgG2b, k Plate 4 C05 CD274 29E.2A3 mouse IgG2b, k Plate 4 C06 CD319 162.1 mouse IgG2b, k Plate 4 C07 CD32 FUN-2 mouse IgG2b, k Plate 4 C08 CD326 9C4 mouse IgG2b, k Plate 4 C09 CD338 5D3 mouse IgG2b, k Plate 4 C10 CD368 9B9 mouse IgG2b, k Plate 4 C11 CD45RA HI100 mouse IgG2b, k Plate 4 C12 CD45RB MEM-55 mouse IgG2b, k Plate 4 D01 CD49e NKI-SAM-1 mouse IgG2b, k Plate 4 D02 CD52 HI186 mouse IgG2b, k Plate 4 D03 CD66a/c/e ASL-32 mouse IgG2b, k Plate 4 D04 CD85h 24 mouse IgG2b, k Plate 4 D05 CD85 GHI/75 mouse IgG2b, k Plate 4 D06 CD86 IT2.2 mouse IgG2b, k Plate 4 D07 CD92 VIM15b mouse IgG2b, k Plate 4 D08 CXCR7 8F11-M16 mouse IgG2b, k Plate 4 D09 Delta Opioid Receptor DOR7D2A4 mouse IgG2b, k Plate 4 D10 Dopamine Receptor D1 L205G1 mouse IgG2b, k Plate 4 D11 EphA2 SHM16 mouse IgG2b, k Plate 4 D12 FcεRlα AER-37 (CRA-1) mouse IgG2b, k Plate 4 E01 GARP 7B11 mouse IgG2b, k Plate 4 E02 CD215 JM7A4 mouse IgG2b, k Plate 4 E03 Lymphotoxin β Receptor 31G4D8 mouse IgG2b, k Plate 4 E04 MRGX2 K125H4 mouse IgG2b, k Plate 4 E05 TMEM8A SA065C3 mouse IgG2b, k Plate 4 E06 CD254 MIH24 mouse IgG2b, k Plate 4 E07 CD318 CUB1 mouse IgG2b, k Plate 4 E08 IgG3, k Isotype Ctrl MG3-35 mouse IgG3, k Plate 4 E09 CD255 CARL-1 Mouse IgG3, k Plate 4 E10 SSEA-4 MC-813-70 Mouse IgG3, k Plate 4 E11 IgM, κ Isotype Ctrl MM-30 Mouse IgM, k Plate 4 E12 Sialyl Lewis X (dimeric) FH6 Mouse IgM, k Plate 4 F01 TRA-1-81 TRA-1-81 Mouse IgM, k Plate 4 F02 CD160 BY55 Mouse IgM, k Plate 4 F03 CD57 HNK-1 Mouse IgM, k Plate 4 F04 CD66b G10F5 Mouse IgM, k Plate 4 F05 TRA-1-60-R TRA-1-60-R Mouse IgM, k Plate 4 F06 IgG1, κ Isotype Ctrl RTK2071 Rat IgG1, k Plate 4 F07 CD115 9-4D2-1E4 Rat IgG1, k Plate 4 F08 CD201 RCR-401 Rat IgG1, k Plate 4 F09 IgG2a, κ Isotype Ctrl RTK2758 Rat IgG2a, k Plate 4 F10 CD120b 3G7A02 Rat IgG2a, k Plate 4 F11 CD210 3F9 Rat IgG2a, k Plate 4 F12 CD267 1A1 Rat IgG2a, k Plate 4 G01 CD294 BM16 Rat IgG2a, k Plate 4 G02 CD49f GoH3 Rat IgG2a, k Plate 4 G03 CD85 MKT5.1 Rat IgG2a, k Plate 4 G04 CD85d 42D1 Rat IgG2a, k Plate 4 G05 IgG Fc M1310G05 Rat IgG2a, k Plate 4 G06 Integrin β7 FIB504 Rat IgG2a, k Plate 4 G07 XCR1 S15046E Rat IgG2a, k Plate 4 G08 Podoplanin NC-08 Rat IgG2a, l Plate 4 G09 IgG2b, κ Isotype Ctrl RTK4530 Rat IgG2b, k Plate 4 G10 CD132 TUGh4 Rat IgG2b, k Plate 4 G11 CD195 J418F1 Rat IgG2b, k Plate 4 G12 CX3CR1 2A9-1 Rat IgG2b, k Plate 4 H01 IgM, κ Isotype Ctrl RTK2118 Rat IgM, k Plate 4 H02 SSEA-3 MC-631 Rat IgM, k Plate 4 H03 CD177 MEM-166 mouse IgG1, k Plate 4 H04 IL1RAP 89412 mouse IgG1, k
TABLE-US-00003 TABLE 3 Antibodies and Reagents Catalog Name of Product Target Fluorochrome Company Isotype # LEGENDScreen.sup.a SCREEN PE BioLegend 10 different 700001 Human PE Kit LEGENDScreen.sup.a SCREEN PE BioLegend 10 different 700007 Human PE Kit PE/Cy7 anti-human CD3 CD3 PE-Cy7 BioLegend mouse 300316 Antibody IgG2a, k APC/Cy7 anti-human CD19 APC-Cy7 BioLegend mouse 302218 CD19 Antibody IgG1, k Alexa Fluor{umlaut over ( )} 488 anti- CD34 AF488 BioLegend mouse 343518 human CD34 Antibody IgG1, k APC/Cy7 anti-human CD34 APC-Cy7 BioLegend mouse 343614 CD34 Antibody IgG2a, k BV421 Mouse Anti- CD34 BV421 BD mouse 562577 Human CD34 IgG1, k BV711 Mouse Anti- CD38 BV711 BD mouse 563965 Human CD38 Clone IgG1, k HIT2 (RUO) PE-Cy7 Mouse Anti- CD45RA PE-Cy7 BD mouse 560675 Human CD45RA IgG2b, k Alexa Fluor 488 anti- CD123 AF488 BioLegend mouse 306036 human CD123 Antibody IgG1, k PerCP/Cyanine5.5 anti- CD90 PerCP-Cy5.5 BioLegend mouse 328117 human CD90 (Thy1) IgG1, k Antibody APC anti-human CD45 CD45 APC BioLegend mouse 982304 Antibody IgG1, k PE anti-human CD177 CD177 PE BioLegend mouse 315806 Antibody IgG1, k Human IL-1 RAcP/IL-1 IL1RAP PE RnD mouse FAB676P R3 PE-conjugated Systems IgG1, k Antibody PE anti-human CD62E CD62E PE BioLegend mouse 336008 Antibody IgG1, k PE anti-human CD54 CD54 PE BioLegend mouse 353105 Antibody IgG1, k PE anti-human CD244 CD244 PE BioLegend mouse 393507 (2B4) Antibody IgG1, k PE anti-human Jagged 2 Jagged PE BioLegend mouse 346904 Antibody 2 IgG1, k PE anti-human CD323 CD323 PE BioLegend mouse 356703 (JAM3) Antibody IgG2a, k PE anti-CD105 CD105 PE BioLegend mouse 800503 (Endoglin) Antibody IgG1, k PE anti-human CD369 CD369 PE BioLegend mouse 355403 (Dectin-1/CLEC7A) IgG2a, k Antibody PE anti-human CD352 SLAMF6 PE BioLegend mouse 317207 (NTB-A) Antibody IgG1, k Anti-SLAMF6 antibody SLAMF6 — Abcam mono IgG ab238421 [EPR22170] rabbit PE Mouse IgG1, κ ISO PE BioLegend mouse 400112 Isotype Ctrl Antibody IgG1, k PE Mouse IgG2a, κ ISO PE BioLegend mouse 400214 Isotype Ctrl (FC) IgG2a Antibody Rabbit IgG, monoclonal ISO — Abcam mono IgG ab199376 [EPR25A] rabbit 7-AAD Viability Staining 7AAD — BioLegend — 420404 Solution 4′,6-Diamidine-2′- DAPI — Sigma- — D9542 phenylindole Aldrich dihydrochloride
TABLE-US-00004 TABLE 4 Top Cell Surface Marker Candidates Rank NBM#1 NBM#2 NBM#3 AML#48 AML#83 AML#80 AML − Rank Mean Rank Specificity Gene Symbol MFI MFI MFI MFI MFI MFI NBM AML/NBM Rank 1 CD123 IL3RA 86.7 1000 1059 2893 4057 1121 3 12 7.5 2 CD369 CLEC7A 1106 88.7 2650 1920 15 1 8 3 CD352 SLAMF6, NTB-A 153 689 627 1754 1495 3253 14 6 10 4 CD54 ICAM1 −427 1302 1134 633 4308 8953 2 18 10 5 CD105 ENG 320 1764 1154 1199 3678 6067 6 14 10 6 CD370 CLEC9A 33.3 653 1197 241 2025 4185 18 4 11 7 CD323 JAM3 1384 1067 3292 3345 7 24 15.5 8 CD93 CD93 140 1381 1906 1890 1784 5837 9 22 15.5 9 CD11c ITGAX −1528 988 550 −249 1206 3379 19 15 17 10 TRA-2-54 ALPL 692 465 4091 1686 26 8 17 11 IL1RAP IL1RAP −53.3 564 305 440 1818 1655 22 20 21 12 CD252 TNFSF4 −300 194 42.7 4159 1931 35 7 21 13 CD261 TNFRSF10A −414 1130 434 −849 2609 2359 16 33 24.5 14 CD56 NCAM1 −80 587 302 19.4 3138 1165 49 13 31 15 CD49d ITGA4 1295 2558 3163 2574 2495 7646 20 43 31.5 16 TLT-2 TRML2 −883 646 420 7.77 1621 1567 44 21 32.5 17 CD11b ITGAM −541 758 358 34.9 1784 1931 36 31 33.5 18 CD314 KLRK1 −501 1251 2231 −793 2911 4049 13 55 34 19 CD49c ITGA3 −681 1244 1774 −2513 2442 3525 30 44 37 20 CD266 TNFRSF12A −1309 641 64.2 73.8 694 602 77 5 41 21 CD165 ADAMTSL1 60 1448 1902 303 4281 2578 62 23 42.5 22 CD344 FZD4 −675 2283 1392 −745 3040 4116 56 41 48.5 23 Jagged 2 JAG2 −7099 2676 1561 637 4057 3785 5 93 49 24 CD7 CD7 −387 661 676 6668 1667 1183 41 58 49.5 25 CD156c ADAM10 856 3189 4176 3403 4903 4352 12 88 50 26 CD354 TREM1 40 1280 567 562 1392 2168 81 27 54 27 CD94 KLRD1 −2968 383 516 −42.7 851 1136 71 38 54.5 28 CD263 TNFRSF10C −340 1523 903 −233 2505 2262 43 69 56 29 CD124 IL4R 127 1310 1152 507 3145 1658 83 29 56 30 CD1d CD1D −795 1327 136 −813 2231 2609 47 67 57 31 CD144 CDH5 133 1106 1138 −350 1892 3641 54 60 57 32 Siglec-8 SIGLEC8 −2353 1478 59.1 −315 2099 1788 10 105 57.5 33 PSMA FOLH1 −133 817 705 50.5 1532 1376 65 53 59 34 CD134 TNFSRF4 40 982 943 284 1529 1856 74 45 59.5 35 CD258 TNFSRF14 −73.3 1874 53.9 −483 2177 1518 119 3 61 36 TCR α/β TRAC −3013 1227 939 159 1670 1798 51 72 61.5 37 CD275 ICOSLG 80 1278 722 491 1822 1735 76 48 62 38 CD182 CXCR2 180 483 438 745 1002 −594 82 42 62 39 SSEA-5 −1099 1418 152 117 2095 2414 27 99 63 40 CD45RB PTPRC 153 395 151 503 1957 500 109 17 63 41 Siglec-10 SIGLEC10 −748 1424 1290 −257 2266 3234 52 79 65.5 42 MICA/MICB MICA/MICB −534 1076 504 −3.88 1199 1337 79 52 65.5 43 IgD IGHD −2154 978 661 −11.6 1358 21 113 67 44 TCR γ/δ −3825 1964 1332 −307 2975 2283 40 94 67 45 CD36L1 SCARB1 1072 974 1600 2128 80 54 67 46 TRA-2-49 ALPL 1405 884 1950 1758 75 64 69.5 47 CD269 TNFRSF17 614 406 958 1360 112 28 70 48 CD304 NRP1 −80 283 275 54.4 862 593 117 25 71 49 CD244 SLAMF4, 2B4 1817 4008 3077 4077 2461 5676 4 139 71.5 50 CD51 ITGAV −86.7 457 370 −1099 1888 605 135 10 72.5
[0167] Flow Cytometry Target Validation
[0168] Protein expression of the top candidates from the screen was confirmed using separate flow cytometry analyses of mononuclear cells from AML and NBM bone marrow samples. Analyses were performed on an LSR Fortessa (BD Bioscience, USA), corresponding isotype controls were used to determine positive cells. All antibodies and reagents used are listed in Table 3.
[0169] Antibody Dependent Cellular Cytotoxicity
[0170] Antibody dependent cellular cytotoxicity (ADCC) assays were performed as previously described in Landberg et al., 2018.sup.24. Target cells were labeled with the membrane dye PKH26 (Sigma-Aldrich, USA) and subsequently incubated with antibodies of varying concentrations for 30 minutes. Freshly isolated or frozen NK cells from healthy donors were then added in a 10:1 ratio compared to target cells. Corresponding isotype antibodies and wells with only NK and target cells were used as controls. The ADCC effect was assessed by flow cytometry after 12-18 hours using an LSR Fortessa (BD Bioscience, USA), with the viability dye DAPI (Sigma-Aldrich, USA) and CountBright Absolute Counting Beads (Thermo Fisher Inc, USA) added to each well. Specific ADCC-induced cell death was calculated with the formula: percentage viable cells.sup.antibody/percentage viable cells.sup.no antibody, and percentage viable cell.sup.isotype/percentage viable cells.sup.no antibody respectively.
[0171] AML Xenografts
[0172] Viably frozen mononuclear cells from bone marrow of AML patients were thawed and T cell depleted using CD3 microbead separation (Miltenyi Biotec) or OKT3 anti-CD3 antibody (BioXCell). For primary and secondary transplantations, ≥5 million cells were transplanted by tail vein injection to sublethally irradiated (250 cGy) NOD.Cg-Prkdc.sup.scidII2rg.sup.tm1WjI/SzJ-SGM3 (NSGS) mice, a variant of the NSG mouse overexpressing hGM-CSF, hIL-3 and hSCF (Jackson laboratory). Mice were euthanized upon signs of serious illness. In vivo experiments were approved by the regional Animal Ethics Committee of Malmö/Lund.
[0173] Statistical Analyses
[0174] Statistical tests were performed using Prism 6 (GraphPad Software, USA). Students T-test or Mann-Whitney U test was used when comparing two groups. Spearman's rank test was used to determine correlations between biological replicates when conducting the antibody screen.
[0175] Results
[0176] Antibody Based Screen Identifies Multiple Candidate Cell Surface Markers
[0177] To identify new cell surface markers specifically expressed on immature AML cells, an arrayed antibody screening system was used to evaluate 362 different cell surface markers within the immature 7AAD.sup.−CD3.sup.−CD19.sup.−CD34.sup.+CD38.sup.− fraction of TP53 mutated AML bone marrow and NBM controls (
[0178] Flow Cytometric Validation Show Overexpression of SLAMF6 in TP53 Mutated AML Samples
[0179] Based on the antibody screen, eight novel markers were chosen for further validation (
[0180] SLAMF6 is Expressed on Immature TP53 AML Cells
[0181] To further delineate SLAMF6 expression in TP53 mutated AML, different cellular compartments were examined in AML samples with retained CD34/CD38 phenotypic hierarchies. All three TP53 mutated AML samples showed high SLAMF6 expression in the immature CD34.sup.+CD38.sup.− compartment as compared to the more mature CD34.sup.− compartments (
[0182] SLAMF6 is Expressed in AMLs of Diverse Genetic Subtypes
[0183] Given that all tested TP53 mutated AML samples showed SLAMF6 expression, the gene expression levels of SLAMF6 in MNC were investigated in the publicly available TCGA data set..sup.32 A higher mean expression in TP53 mutated (n=14) compared to wild type AML (n=151) was observed (p=0.001,
[0184] SLAMF6 Antibody Induces ADCC Mediated Killing of KG1 Cells
[0185] To evaluate SLAMF6 as a target for antibody-based therapies, a series of AML cell lines were first investigated for expression of SLAMF6. KG1 and K562 cells both expressed high levels of SLAMF6, while OCI-AML 3 showed no expression (
[0186] SLAMF6 Expression is Retained after Serial Xenografting
[0187] To establish a disease model allowing in vivo studies for antibody-based targeting of SLAMF6, primary AML samples were transplanted to sublethally irradiated NSGS mice. Three TP53 mutated AML samples were serially transplanted and two of these (AML 48 and AML 80) showed high leukemic engraftment in secondary mice. SLAMF6 expression was highly retained in both of these samples, showing the feasibility of studying SLAMF6 in vivo (
[0188] Discussion
[0189] To improve the survival of patients with neoplastic hematologic disorders (including AML), a better understanding of the disease- and relapse-causing leukemic stem cells and possibilities to specifically target such cells are needed. By identifying cell surface markers specifically expressed on AML stem cells, their prospective isolation for functional interrogation becomes feasible. Such cell surface markers may also provide attractive targets for directed treatments as shown for several markers including CD33, CD123 and IL1RAP..sup.9, 14, 33 Because AML is a heterogeneous disease both in terms of underlying molecular cause and response to therapy, searching for cell surface markers in specific genetic subtypes of AML might increase the chance of identifying such markers. This in turn could provide specific biological insights into AML subtypes with treatment implications in parity with ATRA treatment for t(15;17) acute promyelocytic leukemia..sup.34 In this study, TP53 mutated AML was a focus, which is one of the subtypes recognized by European Leukemia Net as having the worst prognosis of all AML subtypes..sup.35 Using an arrayed antibody screen of 362 cell surface markers, specifically upregulated markers were screened for on CD3.sup.−CD19.sup.−CD34.sup.+CD38.sup.− AML cells compared to corresponding cells from normal bone marrow. Using this approach, several previously described markers were identified including CD123, IL1RAP and CD93, thus validating our screening approach. Importantly, SLAMF6 was identified as a new marker being upregulated on immature TP53 mutated AML cells and SLAMF6 antibodies were showed that can recruit human NK cells to elicit cell killing of AML cells.
[0190] SLAMF6 is one of nine members of the SLAM family of paralogue genes located on chromosome band 1q23, most of which play a role in immune regulation and some that have been suggested as therapeutic targets in different malignancies..sup.36 Elotuzumab is a naked antibody targeting SLAMF7 that has been shown to both mark myeloma cells for effector cell mediated killing and induce an immune response against the myeloma cells through the antibody's activating effect upon binding to normal NK cells..sup.37, 38 This dual mode of action immune therapy is a promising, novel treatment concept. Elotuzumab has shown promising effects in clinical trials for treatment of myeloma..sup.39 SLAMF6 is known to be expressed on human B, T and NK cells. Upon homophilic self-ligation of SLAMF6, internal signaling through tyrosine phosphorylation of SLAMF6 cytoplasmic tail, recruitment of SAP or EAT-2 is involved in NK cell and T cell activation..sup.4041 SLAMF6 also plays a role in T cell exhaustion and an anti-SLAMF6 antibody was shown to reactivate exhausted CD8.sup.+ T cells, another potential antineoplastic effect that targeting SLAMF6 with an antibody could elicit..sup.42 However, SLAMF6 can also inhibit cellular functions through recruitment of SHP-1/2 in the absence of SAP, making the exact effects of SLAMF6 signaling or binding context- and cell-dependent..sup.43 In the present study, SLAMF6 was found to be upregulated in the immature CD34.sup.+CD38.sup.− subpopulation of AML cells, which in most subtypes of AML has been shown to contain the highest AML stem cell activity as measured by long-term engraftment in immunodeficient mice..sup.15 Notably, SLAMF6 was not expressed on immature normal CD34.sup.+CD38.sup.− bone marrow cells, suggesting that directed therapies against SLAMF6 would spare normal hematopoietic stem cells. SLAMF6 expression was however retained in CD3.sup.+ T cells and CD19.sup.+ B-cells in all AML samples analyzed, independent of genetic alterations in the AML sample. KG1 cells were also shown to express high levels of SLAMF6 and these cells were specifically killed in ADCC experiments using an anti-SLAMF6 antibody. Importantly, SLAMF6 was retained on AML cells after serial transplantation to NSGS mice.
[0191] In conclusion, SLAMF6 was identified as a cell surface marker upregulated on immature AML cells, for example those carrying a TP53 mutation. SLAMF6 was further demonstrated to provide a new target for antibody-based therapies in AML, thus opening up new avenues for the development of antibody-based therapeutic strategies for AML, including those subtypes with poor prognosis (such as TP53 mutated AML). Further, although the cell death mechanism of action demonstrated in this experiment is ADCC, the induction of cell death would be achievable with other mechanisms of action based on this finding that SLAMF6 is present on the immature AML cells. For example, SLAMF6 could be targeted with an antibody that comprises a radiolabel or cytotoxic moiety.
EXAMPLE 2
[0192] SLAMF6 Antibody Induces ADCC of AML Patient Cells
[0193] Summary
[0194] SLAMF6 antibodies can induce ADCC to kill AML patient cells.
[0195] Introduction
[0196] Killing of cancer cells through ADCC with a SLAMF6 antibody has never before been demonstrated. Here, it is demonstrated that SLAMF6-expressing primary AML patient samples and xenografted AML patient samples enriched for leukemia stem cells can be killed ex vivo by ADCC using SLAMF6 antibodies.
[0197] Materials and Methods
[0198] AML Patient Samples
[0199] Bone marrow and peripheral blood samples from AML patients were collected at the Department of Clinical Genetics, Skene University Hospital after written informed consent. Mononuclear cells were prepared by lymphoprep separation (GE Healthcare) and viably frozen. Protein expression of SLAMF6 on the leukemia cells was determined by flow cytometry with a SLAMF6 antibody (Biolegend).
[0200] Patient-Derived Xenografts
[0201] To generate patient-derived xenografts, primary AML patient cells were thawed, and T cells depleted by either CD3 microbead separation (Miltenyi Biotec) or treatment with the OKT3 anti-CD3 antibody (BioXCell). A total of ≥5 million cells were then transplanted by tail vein injection to sublethally irradiated NOD.Cg-Prkdc.sup.scidII2rg.sup.tm1WjI/SzJ-SGM3 (NSG-S) mice (250 cGy), a substrain of the NSG mouse overexpressing hGM-CSF, hIL-3 and hSCF (Jackson laboratory). Mice were euthanized upon signs of serious illness.
[0202] Antibody Dependent Cellular Cytotoxicity
[0203] ADCC assays were performed as described in Example 1. Target cells were labeled with the membrane dye PKH26 (Sigma-Aldrich, USA) and subsequently incubated with rabbit monoclonal SLAMF6 antibody or an isotype control (Biolegend) for 30 minutes. Freshly isolated NK cells from healthy donors were then added in a 10:1 ratio compared to target cells. Corresponding isotype antibodies and wells with only NK and target cells were used as controls. The ADCC effect was assessed by flow cytometry after 12-18 hours using an LSR Fortessa (BD Bioscience), with the viability dye DAPI (Sigma-Aldrich) and CountBright Absolute Counting Beads (Thermo Fisher Inc) added to each well.
[0204] Results
[0205] Treatment with a SLAMF6 antibody induced cell death in AML patient cells ex vivo by recruitment of NK effector cells and killing through ADCC (
[0206] Discussion
[0207] These data show that an antibody against SLAMF6 can elicit killing of AML patient cells and of leukemia stem cell-enriched AML samples ex vivo, by binding to the target cell and recruiting effector cells to induce ADCC. This demonstrates that SLAMF6 antibodies have therapeutic activity against AML patient cells.
EXAMPLE 3
[0208] SLAMF6 expression on AML stem cells and AML cell lines
[0209] Summary
[0210] SLAMF6 is expressed on leukemic stem cells in a majority of AML patients in AML of diverse genetic background.
[0211] Introduction
[0212] To determine the relevance of SLAMF6 as a target for therapy in AML, SLAMF6 protein expression was investigated in a cohort of 42 primary AML patient samples and nine AML cell lines. The expression was further studied in the leukemic stem cell containing compartment with a CD3.sup.−CD19.sup.−CD34.sup.+CD38low immunophenotype.
[0213] Materials and Methods
[0214] Bone marrow and peripheral blood samples were collected after written informed consent in accordance to the Declaration of Helsinki. Samples were collected from patients with AML and myelodysplastic syndrome (MDS). Mononuclear cells (MNC) were isolated using Lymphoprep (GE Healthcare Bio-Sciences AB, Sweden) and subsequently viably frozen. Patients included in the study and their clinical characteristics are shown in Table 5. The study was approved by a regional ethics committee in Lund (Dnr 2011/289). SLAMF6 expression was determined by flowcytometry on an LSR Fortessa (BD Bioscience, USA) with commercially available antibodies targeting CD3, CD19, CD34, CD38 and SLAMF6 as well as a viability marker.
[0215] Results
[0216] A cohort of 42 primary AML samples was evaluated for SLAMF6 expression. For CD34 expressing AML samples, the CD34+ and the CD34+CD38low cells within the CD3−CD19− compartment known to be enriched for leukemic stem cells were specifically evaluated. Samples were classified as high (“SLAMF6high”) when >50% of cells expressed SLAMF6, intermediate (“SLAMF6int”) when 10-50% of cells expressed SLAMF6 and negative (“SLAMF6neg”) when <10% of cells expressed SLAMF6. Within the CD3−CD19− myeloid compartment, 26% of AML samples were classified as SLAMF6high and 33% as SLAMF6int, within the CD34+ compartment 45% were classified to be SLAMF6high and 21% SLAMF6int and within the CD34+CD38low compartment 41% were SLAMF6high and 24% were SLAMF6int (
[0217] Discussion
[0218] SLAMF6 is shown to be aberrantly expressed on leukemic stem cells from primary AML samples carrying a large variety of genetic alterations.
TABLE-US-00005 TABLE 5 AML Patient Characteristics CD34+ Pat# Gender Disease FAB ELN Karyotype Mutations CD34% Myeloid CD34+ CD38low 7 F tAML M4 tAML IDH2, FLT3, KRAS, 1 Int — — diagnosis NPM1, WT1, DNMT3A 9 F AML M5 LR 46, XX DNMT3A, TET2, NPM1 0 Neg — — diagnosis 10 M sAML M4 sAML 46, XY, inv(9)(p11q12) RUNX1, ASXL1, IDH2, 99 Neg Neg Neg diagnosis JAK2, SRSF2, 21 M AML M2 IR 46, XY IDH2, STAG2, BCOR 94 Neg Neg Neg diagnosis 23 M AML M4 LR 46, XY, der(16)(q22) NRAS 46 Int High High diagnosis 24 M AML M2 LR 46, XY IDH2, NPM1, FLT3-ITD 0 Neg — — diagnosis 25 M AML M2 LR 45, X, −Y NPM1, TET2 5 Neg — — diagnosis 28 M sAML M2 sAML 46, XY NPM1, FLT3-ITD, TET2 1 Neg — — diagnosis 32 M AML M2 HR 47, XY, +13 RUNX1, ASXL1 73 Int Int Int diagnosis 33 F AML M4 sAML 46, XX DNMT3A, NPM1, IDH1, 3 High — — relapse CEBPA 34 F AML M5 IR 46, XX DNMT3A, IDH1, NRAS, 96 High High High diagnosis CEBPA 35 F MDS NA HR 46, XX RUNX1, ASXL1, DNMT3A, 59 Int Int Int RAEB-2 IDH2 37 M AML M5 HR Complex1 TP53, FLT3 2 Int — — diagnosis 48 F tAML M2 tAML Complex2 TP53 63 High High High dignosis 55 M tAML M2 tAML 45, XY, −7/45, X, −Y SRSF2, ASXL1, EZH2, 42 High High High diagnosis PTPN11 61 F AML M2 LR Complex3 RUNX1, ASXL1, RB1 52 High High High diagnosis 62 F AML M4 HR Complex4 TP53, DNMT3A, 15 Neg Neg Neg diagnosis 66 M sAML M2 sAML 46, XY IDH1, NPM1, JAK2, 4 Neg — — diagnosis CEBPA 70 M MPN/MDS NA NA 46, XY RUNX1, ASXL1, TET2 10 Neg Neg Neg 80 M AML M2 HR 48, XXYc, +i(12)(p10), TP53 35 Int High High diagnosis der(17)t(13;17)(q1?4;p1?3) 83 M sAML M2 sAML Complex5 TP53, TET2, NF1 15 Int High High diagnosis 85D F AML M2 HR Complex6 TP53, DNMT3A 60 Neg Neg Neg diagnosis 85R F AML M2 HR N/A TP53, DNMT3A 91 Int Int Int relapse 94 M AML M0 HR 47, XY, +10 DNMT3A, IDH1, FLT3- 98 Neg Neg Neg diagnosis ITD, RUNX1, BCOR, STAG2 97 F AML M4 LR 46, XX NPM1, FLT3-ITD, IDH1, 39 Int Int Int diagnosis DNMT3A 104 M tAML M0 tAML 46, XY NPM1, FLT3-ITD, IDH2, 0 Neg — — diagnosis DNMT3A 105 F AML M4 IR 46, XX NPM1, FLT3-ITD, 10 High High High diagnosis DNMT3A, 110 M AML M2 IR 46, XY IDH2, SRSF2, STAG2, 7 Neg — — diagosis 111 M sAML M2 sAML 47, XY, +8/47, XY, +mar RUNX1, BCOR 4 Neg — — diagnosis 117 M AML M2 LR 46, XY, t(8;21)(q22;q22) KIT, NRAS 57 High High Int diagnosis 123 F AML M4 LR 46, XX, t(16;16)(p13;q22) — 70 High High High diagnosis 124 M AML M4 LR 46, XY, inv(16)(p13q22) KIT 91 High High High diagnosis 126 F AML M1 HR 46, XX IDH2, CBFC, CEBPA, 60 Neg Neg Neg diagnosis RAD21, MEIS1 136 F AML M4 LR 46, XX NPM1, IDH2 4 High — — diagnosis 138 M AML M4 HR 48, XY, +13, +19 RUNX1, TET2, ZRSR2 98 Neg Neg Neg diagnosis 144 F MPN/MDS NA NA 47, XX, +8 RUNX1, ASXL1, TET2, 15 Int Neg Neg BCOR 151 M sAML M1 sAML 46, XY, ?del(Y)(q11) RUNX1, ASXL1, TET2, 96 Neg Neg Neg diagnosis IDH1, SRSF2, PHF6 154 M tAML M0 tAML 46, XY, inv(2)(p21q31), RUNX1, SF3B1, NF1, 20 Int High High diagnosis t(3;12)(q26;p13) PTPN11, ASXL2, IKZF1 155 M AML M5 HR Complex7 TP53, FLT3 2 High — — diagnosis 157 M sAML M4 sAML 46, XY SRSF2, NRAS 49 Int Int Int diagnosis 161 F AML M2 LR 46, XX, t(8;21)(q22;q22) FLT3-ITD, KIT, KRAS, 64 Int Int Int diagnosis RUNX1 172 F AML M4 HR 46, XX, t(3;12)(q26;p13) FLT3-ITD, ASXL1, RUNX1 26 Int High High diagnosis Abbreviations; sAML: secondary AML, tAML: therapy-related AML, ELN: European Leukemia net risk classification 2017, LR: low risk, IR: intermediate risk, HR: high risk Complex1: 47, XY, +6, +i(8)(q10), −18, −22, +mar/45-46, XY, −3, +add(6)(p21), +i(8)(q10), der(16)t(3;16)(p12;q11), −22 Complex2: 43-47, XX, del(5)(q13q33), der(8)t(8;12)(p22;p13), add(11)(p15), −12, add(13)(p11), −18, del(20)(q11), add(22)(q13), +mar Complex3: 45, X, −X, der(8)t(8;21)(q22;q22), del(9)(q13), del(13)(q21q21), del(17)(p?13), der(21)t(8;21)ins(21;?)(q22;?) Complex4: 46, XX, der(3)t(1;3)(p13;q27), −5, add(7)(q22), der(16)t(?5;16)(q?;p11), der(17)t(5;17)(q?;q21), der(21)t(11;21)(q13;q22), +mar Complex5: 45, XY, der(13;14)(q10;q10)/42-44, idem, −5, −7, −9, −10, −11 , ?hsr(11)(q23), −14, −16, −20 , +4mar Complex6: 43-49, XX, −3, add(6)(p21), −13, add(13)(q34), add(17)(p11), +2-5r/46, XX, del(5)(q13q33) Complex7: 47, XY, +8, t(9;11)(p21;q23)/46-47, idem, der(9)del(9)(p12)del(9)(q12), der(17)t(9;17)(q31;p13)/46-47, idem, der(7;10)(q10;q10), +add(8)(p11)
EXAMPLE 4
[0219] Activation of SLAMF6 on T Cells Promotes Killing of AML Cells
[0220] Summary
[0221] Stimulation of T cells with an activating SLAMF6 antibody induces T cell-mediated killing of AML cells.
[0222] Introduction
[0223] SLAMF6 is a self-ligand, binding to other SLAMF6 molecules on the surface of interacting cells. Since SLAMF6 is expressed both on leukemia stem cells and on certain normal immune cells (e.g. T, B and NK cells), modulating these interactions could have therapeutic potential. Therefore, the effect on T cell-mediated killing of AML cells by T cell stimulation was determined with an activating SLAMF6 antibody.
[0224] Materials and Methods
[0225] T cells were isolated by CD3 microbead separation (Miltenyi Biotec) of leukocyte concentrate collected from healthy donors and viably frozen. T cell-mediated killing was assessed by incubating 80,000 T cells and 20,000 HNT-34 target cells with a SLAMF6 antibody or an isotype control (Biolegend) for 72 hours before quantification on an LSR Fortessa (BD Biosciences) with CountBright Absolute Counting Beads (Thermo Fisher) and antibodies against CD3 and CD33 (Biolegend).
[0226] Results
[0227] T cell stimulation with an activating antibody against SLAMF6 markedly increased T cell-mediated killing of AML target cells at all tested concentrations (
[0228] Discussion
[0229] These data show that targeting SLAMF6 on immune cells modulates their response to leukemia and induces T cell-mediated killing of AML cells. A therapeutic agent could thus act either on AML cells, on interacting immune cells or on both cell types in combination, to elicit cell killing of leukemic cells.
EXAMPLE 5
[0230] Knockout of SLAMF6 from AML Cells Promotes T Cell-Mediated Killing
[0231] Summary
[0232] SLAMF6 protects AML cells from T cell-mediated killing. Knocking out SLAMF6 in AML cells promotes T cell expansion, activation and killing of the AML cells.
[0233] Introduction
[0234] The functional importance of SLAMF6 on AML cells was determined by knocking out SLAMF6 in AML cells by CRISPR-Cas9 and analyzing the effect on T cell-mediated killing.
[0235] Materials and Methods
[0236] SLAMF6 knockout cell lines were generated by introduction of Cas9 protein (PNA Bio) and one of two different SLAMF6 gRNA constructs, or a negative control gRNA against luciferase, by electroporation with an ECM 830 Electroporation System (Harvard Apparatus), followed by sorting of successfully transfected cells after 24 h with a FACS Aria (BD Biosciences). Knockout was verified by FACS with a SLAMF6 antibody (Biolegend) before initiation of experiments. T cells were isolated by CD3 microbead separation (Miltenyi Biotec) of leukocyte concentrate collected from healthy donors. T cell activation and T cell-mediated killing were assessed by incubation of 80,000 T cells and 20,000 target cells for 72 hours before quantification on an LSR Fortessa (BD Biosciences) with CountBright Absolute Counting Beads (Thermo Fisher) and antibodies against CD3 and CD33 (Biolegend).
[0237] Results
[0238] Removal of SLAMF6 from KG-1 AML cells by CRISPR-Cas9 (
[0239] Discussion
[0240] This finding demonstrates that SLAMF6 protects leukemia cells against T cell-mediated killing and that targeting of SLAMF6 on AML cells or modulation of the SLAMF6-SLAMF6 interaction between leukemia cells and immune cells promotes an anti-leukemia immune response.
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EMBODIMENT PARAGRAPHS
[0314] Accordingly, the present application also provides aspects according to the following numbered paragraphs:
[0315] 1. An agent comprising or consisting of a binding moiety with specificity for Signaling Lymphocytic Activating Molecule Family Member 6 (SLAMF6) for use in inducing cell death and/or inhibiting the growth and/or proliferation of pathological stem cells and/or progenitor cells associated with a neoplastic hematologic disorder, wherein the cells express SLAMF6.
[0316] 2. An agent comprising or consisting of a binding moiety with specificity for Signaling Lymphocytic Activating Molecule Family Member 6 (SLAMF6) for use in detecting pathological stem cells and/or progenitor cells associated with a neoplastic hematologic disorder, wherein the cells express SLAMF6.
[0317] 3. An agent according to paragraph 1 or paragraph 2 wherein the neoplastic hematologic disorder is a leukemia, optionally wherein [0318] (a) the pathological stem cells are leukemic stem cells; and/or [0319] (b) the progenitor cells are leukemic progenitor cells.
[0320] 4. An agent according to any one of the preceding paragraphs wherein the cells expressing SLAMF6 also express CD34.sup.+CD38.sup.−.
[0321] 5. An agent according to any one of the preceding paragraphs wherein the neoplastic hematologic disorder is associated with cells comprising a TP53 mutation.
[0322] 6. An agent according to paragraph 5 wherein the cells expressing SLAMF6 also express CD34.sup.+CD38.sup.− and wherein the cells comprise a TP53 mutation.
[0323] 7. An agent according to any one of the preceding paragraphs wherein the neoplastic hematologic disorder is selected from the group consisting of chronic myeloid leukemia (CML), myeloproliferative disorders (MPD), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).
[0324] 8. An agent according to any one of the preceding paragraphs wherein the neoplastic hematologic disorder is acute myeloid leukemia (AML).
[0325] 9. An agent according to any one of the preceding paragraphs wherein the binding moiety has specificity for human SLAMF6.
[0326] 10. An agent according to any one of the preceding paragraphs wherein SLAMF6 is localised on the surface of a cell.
[0327] 11. An agent according to any one of the preceding paragraphs wherein the agent is capable of modulating an interaction between an immune cell and leukemic stem cells and/or an immune cell and leukemic cells.
[0328] 12. An agent according to paragraph 11 wherein the immune cells are selected from the group consisting of: B cells, T cells and/or NK cells; preferably wherein the immune cells express SLAMF6.
[0329] 13. An agent according to any one of the preceding paragraphs wherein the agent is capable of killing the pathological stem cells and/or progenitor cells.
[0330] 14. An agent according to paragraph 13 wherein the agent is capable of inducing apoptosis of the stem cells and/or progenitor cells.
[0331] 15. An agent according to paragraph 13 or 14 wherein killing of the cells is induced by antibody-dependent cell-mediated cytotoxicity (ADCC) and/or by a T cell mediated mechanism.
[0332] 16. An agent according to any one of the preceding paragraphs wherein the agent comprises or consists of a polypeptide.
[0333] 17. An agent according to paragraph 16 wherein the agent comprises or consists of an antibody or an antigen-binding fragment thereof with binding specificity for SLAMF6, or a variant, fusion or derivative of said antibody or antigen-binding fragment, or a fusion of a said variant or derivative thereof, which retains the binding specificity for SLAMF6.
[0334] 18. An agent according to paragraph 17 wherein the agent comprises or consists of an antibody or antigen-binding fragment thereof with binding specificity for SLAMF6.
[0335] 19. An agent according to paragraph 18 wherein the agent comprises or consists of an intact antibody.
[0336] 20. An agent according to paragraph 18 wherein the agent comprises or consists of an antigen-binding fragment of an antibody.
[0337] 21. An agent according to paragraph 20 wherein antigen-binding fragment is selected from the group consisting of Fv fragments (e.g. single chain Fv, disulphide-bonded Fv and domain antibodies) and Fab-like fragments (e.g. Fab fragments, Fab′ fragments and F(ab).sub.2 fragments).
[0338] 22. An agent according to any one of paragraphs 17 to 21 wherein the antibody is a recombinant antibody.
[0339] 23. An agent according to any one of paragraphs 17 to 21 wherein the antibody is a monoclonal antibody.
[0340] 24. An agent according to any one of paragraphs 17 to 21 wherein the antibody is a polyclonal antibody.
[0341] 25. An agent according to any one of paragraphs 17 to 24 wherein the antibody or antigen-binding fragment thereof is human or humanised.
[0342] 26. An agent according to any one of paragraphs 1 to 16 wherein the agent comprises or consists of a non-immunoglobulin binding moiety.
[0343] 27. An agent according to any one of paragraphs 1 to 16 wherein the agent comprises or consists of an aptamer.
[0344] 28. An agent according to paragraph 27 wherein the agent comprises or consists of a peptide aptamer.
[0345] 29. An agent according to paragraph 27 wherein the agent comprises or consists of a nucleic acid aptamer.
[0346] 30. An agent according to any one of paragraphs 1 to 15 wherein the agent comprises or consists of a small chemical entity.
[0347] 31. An agent according to any one of the preceding paragraphs further comprising a moiety for increasing the in vivo half-life of the agent.
[0348] 32. An agent according to paragraph 31 wherein the moiety for increasing the in vivo half-life is selected from the group consisting of polyethylene glycol (PEG), human serum albumin, glycosylation groups, fatty acids and dextran.
[0349] 33. An agent according to paragraph 31 or 32 wherein the agent is PEGylated.
[0350] 34. An agent according to any one of the preceding paragraphs further comprising a cytotoxic moiety.
[0351] 35. An agent according to paragraph 34 wherein the cytotoxic moiety comprises or consists of a radioisotope.
[0352] 36. An agent according to paragraph 35 wherein the radioisotope is selected from the group consisting of astatine-211, bismuth-212, bismuth-213, iodine-131, yttrium-90, lutetium-177, samarium-153 and palladium-109.
[0353] 37. An agent according to paragraph 34 wherein the cytotoxic moiety comprises or consists of a toxin (such as saporin or calicheamicin).
[0354] 38. An agent according to paragraph 34 wherein the cytotoxic moiety comprises or consists of a chemotherapeutic agent (such as an antimetabolite).
[0355] 39. An agent according to any one of the preceding paragraphs further comprising a detectable moiety.
[0356] 40. An agent according to paragraph 39 wherein the detectable moiety comprises or consists of a radioisotope.
[0357] 41. An agent according to paragraph 40 wherein the radioisotope is selected from the group consisting of: technetium-99m; indium-111; gallium-67; gallium-68; arsenic-72; zirconium-89; iodine-12; thallium-201.
[0358] 42. An agent according to paragraph 39 wherein the detectable moiety comprises or consists of a paramagnetic isotope.
[0359] 43. An agent according to paragraph 42 wherein the paramagnetic isotope is selected from the group consisting of: gadolinium-157; manganese-55, dysprosium-162, chromium-52; iron-56.
[0360] 44. A pharmaceutical composition comprising an effective amount of an agent as defined in any one of the preceding paragraphs and a pharmaceutically-acceptable diluent, carrier or excipient.
[0361] 45. A pharmaceutical composition according to paragraph 44 adapted for parenteral delivery.
[0362] 46. A pharmaceutical composition according to paragraph 44 adapted for intravenous delivery.
[0363] 47. A kit comprising an agent as defined in any one of paragraphs 1 to 43 or a pharmaceutical composition as defined in any one of paragraphs 44 to 46.
[0364] 48. Use of an agent as defined in any one of paragraphs 1 to 43 in the preparation of a medicament for inducing cell death and/or inhibiting the growth and/or proliferation of pathological stem cells and/or progenitor cells associated with a neoplastic hematologic disorder, wherein the cells express SLAMF6.
[0365] 49. Use of an agent as defined in any one of paragraphs 1 to 43 in the preparation of a diagnostic agent for detecting pathological stem cells and/or progenitor cells associated with a neoplastic hematologic disorder, wherein the cells express SLAMF6.
[0366] 50. Use of an agent as defined in any one of paragraphs 1 to 43 for detecting pathological stem cells and/or progenitor cells associated with a neoplastic hematologic disorder, wherein the cells express SLAMF6.
[0367] 51. The use according to paragraph 48, 49 or 50 wherein the neoplastic hematologic disorder is a leukemia.
[0368] 52. A use according to any one of paragraphs 48 to 51 wherein the neoplastic hematologic disorder is selected from the group consisting of chronic myeloid leukemia (CML), myeloproliferative disorders (MPD), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).
[0369] 53. The use according to paragraph 52 wherein the neoplastic hematologic disorder is acute myeloid leukemia (AML).
[0370] 54. A method for inducing cell death and/or inhibiting the growth and/or proliferation of pathological stem cells and/or progenitor cells associated with a neoplastic hematologic disorder in an individual, comprising the step of administering to the individual an effective amount of an agent as defined in any one of paragraphs 1 to 43, or a pharmaceutical composition as defined in paragraph 44 to 46, wherein the cells express SLAMF6.
[0371] 55. A method according to paragraph 54 wherein the neoplastic hematologic disorder is a leukemia.
[0372] 56. A method for detecting pathological stem cells and/or progenitor cells associated with neoplastic hematologic disorder in an individual, comprising the step of administering to the individual an effective amount of an agent as defined in any one of paragraphs 1 to 43, or a pharmaceutical composition as defined in paragraph 44 to 46 wherein the cells express SLAMF6.
[0373] 57. An in vitro method for diagnosing or prognosing a neoplastic hematologic disorder, the method comprising: [0374] (a) providing a bone marrow or peripheral blood sample of haematopoietic cells from an individual to be tested; [0375] (b) isolating a subpopulation of CD34.sup.+, CD38.sup.− cells from the haematopoietic cells; and [0376] (c) determining whether stem cells, contained within the CD34.sup.+, CD38.sup.− cells, express the cell surface marker SLAMF6; [0377] wherein stem cells that exhibit the cell surface marker profile CD34.sup.+, CD38.sup.− and SLAMF6.sup.+ are indicative of the individual having or developing leukemia [0378] optionally wherein the method also includes a step comprising quantification of levels of immune cells (such as B cells, T cells and/or NK cells), preferably wherein the immune cells express SLAMF6.
[0379] 58. A method according to any one of paragraphs 54 to 57 wherein the neoplastic hematologic disorder is a leukemia.
[0380] 59. A method according to any one of paragraphs 54 to 58 wherein the neoplastic hematologic disorder is selected from the group consisting of chronic myeloid leukemia (CML), myeloproliferative disorders (MPD), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).
[0381] 60. The method according to paragraph 59 wherein the neoplastic hematologic disorder is acute myeloid leukemia (AML).
[0382] 61. An agent for use in medicine substantially as described herein with reference to the description.
[0383] 62. A pharmaceutical composition substantially as described herein with reference to the description.
[0384] 63. Use of an agent substantially as described herein with reference to the description.
[0385] 64. A method of treatment or diagnosis as described herein with reference to the description.
[0386] 65. A kit substantially as defined herein with reference to the description.