A colon cancer survivor-derived antibody recognizes a ......cancer. ‣ The patient B-cell...
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M.Kedde 1,6 , T.Beaumont 1,6 , S.J.Merat 1,6 , M.Kwakkenbos 1 , L.Bartels 1 , D.vd.Berg 1 , Y.B.Claassen 1 , G.Moiset 1 , K.Wagner 1 , A.Q.Bakker 1 , K.Maijoor 1 , M.Bohne 1 , C.Bru 1 , V.Kattler 1 , H.v.Eenennaam 1 , V.H.Roos 2 , F.Kallenberg 2 , J.P.Medema 3 , P.J.Hensbergen 4 , P.v.Helden 1 , E.Dekker 2 and H.Spits 1,5 Background ‣ AT1636 is a fully human antibody, derived from B-cells of a patient with Lynch syndrome who cured metastatic colon cancer. ‣ The patient B-cell repertoire was interrogated for binding to colorectal cancer (CRC) by using AIMMs proprietary B cell immortalization technology[1]. ‣ AT1636 is specific for a previously unidentified O-mannosylated 70kDa E-cadherin variant (ECV). ‣ From the original AT1636 B-cell clone, using the AIMProve method[2], a high-affinity AT1636 IYN variant was generated. ‣ Tumor-specific binding of AT1636 is dependent on the O-mannosylation pattern in the antibody epitope on ECV which was shown to depend on the transmembrane O-mannosyltransferase targeting cadherins 3 (TMTC3)[3]. ‣ A CD3-bispecific antibody (single-chain UCHT1) AT1636 IYN format specifically killed CRC cell lines. Conflict of interest disclosure: MK, TB, SJM, MK, LB, DvB, GM, YBC, GM, AQB, KM, MB, VK, HvE and PvH are employees of AIMM Therapeutics. MK, TB, SJM, MK, LB, DvB, GM, YBC, GM, AQB, KM, MB, VK, HvE, PvH and HS have equity ownerschip in AIMM Therapeutics. references 1) Kwakkenbos, MJ, Diehl, SA, Yasuda, E, Bakker, AQ, van Geelen, CMM, Lukens, MV, van Bleek, GM, et al. Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming. Nature Medicine 2010; 16: 123–128. 2) Wagner, K, kwakkenbos, MJ, Claassen, YB, Maijoor, K, Böhne, M, van der Sluijs, KF, Witte, MD, et al. Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity. Proc Natl Acad Sci USA 2014; 111: 16820–16825. 3) I.S.B. Larsen et al. Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins. Proc Natl Acad Sci USA 2017 1. donor description 3. AT1636 major target is a truncated, mannosylated variant of E-cadherin 4. TMTC3 and E-cadherin co-express in tumor cell- lines (A) TMTC3, a mannosyl-transferases, was described to mannosylate E- cadherin. Shown are number and percentage of tumor cell lines that co- express relative high levels of E- cadherin and TMTC3. https://portals.broadinstitute.org /ccle and J.C. Sunryd, JBC (2014), M.O. Sheikh JBC (2017), M.B. Vester- Christensen PNAS (2013) and I.S.B. Larsen PNAS (2017) (B) TMTC3 and E-cadherin mRNA expression predicts AT1636wt binding by flow cytometry; shown is binding to several colorectal cell lines Conclusion ‣ Mannosylated ECV is a unique novel, potential cancer-specific target recognized by AT1636. ‣ Mannosylated ECV can be found across multiple CRC and other tumor types, its recognition by AT1636 depends on the presence of TMTC3 expression. ‣ Although AT1636 by itself is not able to induce ADCC, the CD3-bispecific AT1636 format specifically killed CRC cell lines. ‣ Overexpression of ECV results in an EMT-like phenotype of the targeted cells (not shown) 5. bispecific T-cell engaging antibody (TCE) from AT1636 and AT1636 IYN TCEs were generated and purified according to methods described by L. Bartels et al. Cancer Res (2019) and L. Bartels et al. Methods (2019) A colon cancer survivor-derived antibody recognizes a previously unidentified truncated, O-mannosylated 70kDa variant of E-cadherin Abstract #4119 AACR: Cancer Therapeutic Targets poster # 5163 A B A B [email protected] Contact us A B 1 AIMM Therapeutics, 2 Department of Gastroenterology & Hepatology and 3 Laboratory for Experimental Oncology and Radiobiology, Center for Experimental and Molecular Medicine, Cancer Center Amsterdam, Amsterdam University Medical Centers, Location AMC, and Oncode Institute, Amsterdam, The Netherlands. 4 Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands. 5 Amsterdam Infection and Immunity Institute (AI&II), Amsterdam, The Netherlands. 6 contribute equally Chemotherapy capecitabine, Avastin and oxaliplatin 2005 2016 Liver resection + radiofrequency ablation Colon resection 2006 2007 Blood obtained with Informed Consent Chemotherapy capecitabine oxaliplatin 5. Production of monoclonal antibodies 4. Screening for high anity antibodies 3.Incubation with CD40L cells and IL-21 2. Transfection 1. Isolation IL-21 CD40L-cells Immortalized B cells Retrovirus carrying Bcl-6 and Bcl-Xl Infected individual Memory B cells No binding observed to: ‣ T cells ‣ fibroblasts ‣ endothelial cells 2. Screening for CRC binding using AIMMs B-cell immortalization technology [1] 0 -10 3 10 3 10 4 10 5 0 20 40 60 80 100 0 -10 3 10 3 10 4 10 5 0 20 40 60 80 100 Normalized To Mode 0 -10 3 10 3 10 4 10 5 0 20 40 60 80 100 Normalized To Mode LS174T DLD-1 COLO205 ‣ Supernatants of memory B cells were screened for binding to 3 CRC cell lines (indicated above). ‣ AT1636 was discovered as an IgG3 (VH3-15 and VK4-1) with 10 amino acid substitutions in the heavy and 9 in the light chain. ‣ AT1636 was produced recombinant as an IgG1 in CHO. ‣ Using AIMProve[2], a high affinity variant has been generated — AT1636IYN. ‣ AT1636 binds cancer cell lines including upper-(aero)digestive tract, esophagus, breast, colon, prostate, pancreas, stomach, urinary tract, ovary and lung cells. A) AT1636 immunoprecipitation from DLD1, reveales a truncated form of E-cadherin as its major target (by mass spectrometry). ‣ The lower band (~70kDa) corresponds to E-cadherin, lacking peptides from the outermost N-terminus (< full length E- cadherin). ‣ The upper band (~90kDa) corresponds to β-catenin, an intracellular protein that interacts with E-cadherin. p70 full length > input AT1636 FT AT1636 IP immunoprecipitation mannose can elute the 70kDa protein from AT1636 > (B) E-cadherin is known to be O-mannosylated [3] ; the AT1636 epitope consists of O-mannosylated residues since it can be eluted with high level mannose. Western blot shows the flowthrough (FT) after an AT1636 IP and the specific elution of p70 (IP) with high levels of mannose 0 5 10 15 6 7 8 9 CDH1 TMTC3 CRC cell lines DLD1 KM12 SW48 HCT116 SW948 COLO205 MDST8 HUTU80 SW480 HT29 CACO2 0 2 4 6 8 10 Caco2 HT29 SW480 HUTU-80 MDST8 Colo205 SW948 HCT116 SW48 KM12 DLD1 AT1636 binding relaBve to control Ab CMS1 CMS2 CMS3 CMS4 unknown ‣ Donor: male with Lynch syndrome (MSH6 gene mutation; CMS-1) ‣ Patient was diagnosed with Stage IV colon carcinoma with liver metastases ‣ Patient is an elite survivor and is tumor free up to today S+C Knob-into-hole mutations sortase-tag on only 1 HC No FcgR interaction bivalent binding to 70kDa mannosylated E-cadherin ! avidity effect Monovalent binding to CD3 ! no avidity effect - AT1636 IYN has an EC50 of ~2 nM by FACS - Low level of kill of A375 is observed which correlates with weak FACS binding (not shown) COLON 62 26% LOW 74% HIGH 44 22.73% LOW 77.27% HIGH PANCREAS 39 43.59% LOW 56.41% HIGH STOMACH 27 96.30% HIGH OESOPHAGUS 3.70% LOW 59 BREAST 37.29% LOW 62.71% HIGH LUNG 191 48.17% LOW 51.83% HIGH 28 60.71% HIGH ENDOMETRIUM 39.29% LOW 39 43.59% LOW 56.41% HIGH URINARY_TRACT 8 100.00% HIGH PROSTATE 54 37.04% HIGH OVARY 62.96% LOW 32 90.63% HIGH UPPER AERODIGESTIVE TRACT 9.38% LOW M Jurkat DLD1 x Jurkat DLD1 x AT1636 Jurkt DLD1 < AT1002 isotype ctrl (40 μg) AT1636 (40 μg) Input (~15 μg) Coomassie (total proteins) < < negative control: grey filed histogram. AT1636: solid black line. 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 0 20 40 60 80 100 concentration [M] specific lysis [%] DLD1 10-12 10-11 10-10 10-9 10-8 10-7 concentration [M] HCT116 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 concentration [M] A375 AT1002 TCE anti-HA Influenza; negative control AT1636wt TCE AT1636IYN TCE TCE positive control EC ₅₀ : 139 pM Max kill: 89 % EC ₅₀ : 476 pM Max kill: 69 % EC ₅₀ : 926 pM Max kill: 91 % 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 concentration [M] HT29 EC ₅₀ : 11 nM Max kill: 12 %
Transcript of A colon cancer survivor-derived antibody recognizes a ......cancer. ‣ The patient B-cell...
M.Kedde1,6, T.Beaumont1,6, S.J.Merat1,6, M.Kwakkenbos1, L.Bartels1, D.vd.Berg1, Y.B.Claassen1, G.Moiset1, K.Wagner1, A.Q.Bakker1, K.Maijoor1, M.Bohne1, C.Bru1, V.Kattler1, H.v.Eenennaam1, V.H.Roos2, F.Kallenberg2, J.P.Medema3, P.J.Hensbergen4, P.v.Helden1, E.Dekker2 and H.Spits1,5
Background
‣ AT1636 is a fully human antibody, derived from B-cells of a patient with Lynch syndrome who cured metastatic colon cancer.
‣ The patient B-cell repertoire was interrogated for binding to colorectal cancer (CRC) by using AIMMs proprietary B cell immortalization technology[1].
‣ AT1636 is specific for a previously unidentified O-mannosylated 70kDa E-cadherin variant (ECV).‣ From the original AT1636 B-cell clone, using the AIMProve method[2], a high-affinity AT1636IYN variant was generated.‣ Tumor-specific binding of AT1636 is dependent on the O-mannosylation pattern in the antibody epitope on ECV which was
shown to depend on the transmembrane O-mannosyltransferase targeting cadherins 3 (TMTC3)[3].‣ A CD3-bispecific antibody (single-chain UCHT1) AT1636IYN format specifically killed CRC cell lines.
Conflict of interest disclosure: MK, TB, SJM, MK, LB, DvB, GM, YBC, GM, AQB, KM, MB, VK, HvE and PvH are employees of AIMM Therapeutics. MK, TB, SJM, MK, LB, DvB, GM, YBC, GM, AQB, KM, MB, VK, HvE, PvH and HS have equity ownerschip in AIMM Therapeutics.
references1) Kwakkenbos, MJ, Diehl, SA, Yasuda, E, Bakker, AQ, van Geelen, CMM, Lukens, MV, van Bleek, GM, et al. Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming. Nature Medicine 2010; 16: 123–128.2) Wagner, K, kwakkenbos, MJ, Claassen, YB, Maijoor, K, Böhne, M, van der Sluijs, KF, Witte, MD, et al. Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity. Proc Natl Acad Sci USA 2014; 111: 16820–16825.3) I.S.B. Larsen et al. Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins. Proc Natl Acad Sci USA 2017
1. donor description
3. AT1636 major target is a truncated, mannosylated variant of E-cadherin
4. TMTC3 and E-cadherinco-express in tumor cell-lines
(A) TMTC3, a mannosyl-transferases, was described to mannosylate E-cadherin. Shown are number and percentage of tumor cell lines that co-express relative high levels of E-cadherin and TMTC3.https://portals.broadinstitute.org/ccleand J.C. Sunryd, JBC (2014), M.O. Sheikh JBC (2017), M.B. Vester-Christensen PNAS (2013) and I.S.B. Larsen PNAS (2017)
(B) TMTC3 and E-cadherin mRNA expression predicts AT1636wt binding by flow cytometry; shown is binding to several colorectal cell lines
Conclusion
‣ Mannosylated ECV is a unique novel, potential cancer-specific target recognized by AT1636.‣ Mannosylated ECV can be found across multiple CRC and other tumor types, its recognition by
AT1636 depends on the presence of TMTC3 expression.‣ Although AT1636 by itself is not able to induce ADCC, the CD3-bispecific AT1636 format
specifically killed CRC cell lines.‣ Overexpression of ECV results in an EMT-like phenotype of the targeted cells (not shown)
5. bispecific T-cell engaging antibody (TCE) from AT1636 and AT1636 IYN
TCEs were generated and purified according to methods described by L. Bartels et al. Cancer Res (2019) and L. Bartels et al. Methods (2019)
A colon cancer survivor-derived antibody recognizes a previously unidentified truncated, O-mannosylated 70kDa variant of E-cadherin
Abstract #4119AACR: Cancer Therapeutic Targetsposter # 5163
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Contact us
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1 AIMM Therapeutics, 2 Department of Gastroenterology & Hepatology and 3 Laboratory for Experimental Oncology and Radiobiology, Center for Experimental and Molecular Medicine, Cancer Center Amsterdam, Amsterdam University Medical Centers, Location AMC, and Oncode Institute, Amsterdam, The Netherlands. 4 Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands. 5 Amsterdam Infection and Immunity Institute (AI&II), Amsterdam, The Netherlands. 6 contribute equally
Chemotherapycapecitabine, Avastin and oxaliplatin
2005 2016
Liver resection + radiofrequency ablationColon resection
2006 2007
BloodobtainedwithInformed Consent
Chemotherapycapecitabine
oxaliplatin
5. Production of monoclonal antibodies
4. Screening forhigh a!nity antibodies
3.Incubation with CD40L cells and IL-21
2. Transfection
1. Isolation
IL-21CD40L-cells
Immortalized B cells
Retrovirus carrying Bcl-6 and Bcl-Xl
Infected individual
Memory B cells
Functional screening(monitor level of infection)
D25 antibody
No binding observed to:‣ T cells‣ fibroblasts‣ endothelial cells
2. Screening for CRC binding using AIMMs B-cell immortalization technology [1]
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eLS174T DLD-1 COLO205
‣ Supernatants of memory B cells were screened for binding to 3 CRC cell lines (indicated above).‣ AT1636 was discovered as an IgG3 (VH3-15 and VK4-1) with 10 amino acid substitutions in the
heavy and 9 in the light chain.‣ AT1636 was produced recombinant as an IgG1 in CHO.‣ Using AIMProve[2], a high affinity variant has been generated — AT1636IYN.‣ AT1636 binds cancer cell lines including upper-(aero)digestive tract, esophagus, breast, colon, prostate,
pancreas, stomach, urinary tract, ovary and lung cells.
A) AT1636 immunoprecipitation from DLD1, reveales a truncated form of E-cadherin as its major target (by mass spectrometry).‣ The lower band (~70kDa) corresponds to E-cadherin, lacking
peptides from the outermost N-terminus (< full length E-cadherin).
‣ The upper band (~90kDa) corresponds to β-catenin, an intracellular protein that interacts with E-cadherin.
p70
full length
>
inputAT16
36 FTAT16
36 IP
immunoprecipitation
mannose can elute the 70kDa protein from AT1636
>
(B) E-cadherin is known to be O-mannosylated[3]; the AT1636 epitope consists of O-mannosylated residues since it can be eluted with high level mannose.Western blot shows the flowthrough (FT) after an AT1636 IP and the specific elution of p70 (IP) with high levels of mannose
0 5 10 156
7
8
9
CDH1
TMTC3
CRCcelllines DLD1
KM12
SW48
HCT116
SW948
COLO205
MDST8
HUTU80
SW480
HT29CACO2
0 2 4 6 8 10
Caco2HT29
SW480HUTU-80MDST8Colo205SW948HCT116SW48KM12DLD1
AT1636bindingrelaBvetocontrolAb
CMS1CMS2CMS3CMS4unknown
‣ Donor: male with Lynch syndrome (MSH6 gene mutation; CMS-1)
‣ Patient was diagnosed with Stage IV colon carcinoma with liver metastases
‣ Patient is an elite survivor and is tumor free up to today
S+C
Knob-into-hole mutations
sortase-tag on only 1 HC
No FcgRinteraction
bivalent binding to 70kDa mannosylatedE-cadherin ! avidity effect
Monovalent binding to CD3 ! no avidity effect
- AT1636IYN has an EC50 of ~2 nM by FACS- Low level of kill of A375 is observed which correlates with weak FACS binding
(not shown)
COLON
62
26% LOW74% HIGH
44
22.73% LOW77.27% HIGH
PANCREAS
39
43.59% LOW56.41% HIGH
STOMACH27
96.30% HIGH
OESOPHAGUS
3.70% LOW
59BREAST
37.29% LOW
62.71% HIGH
LUNG191
48.17% LOW51.83% HIGH
28
60.71% HIGH
ENDOMETRIUM
39.29% LOW
39
43.59% LOW56.41% HIGH
URINARY_TRACT
8
100.00% HIGH
PROSTATE54
37.04% HIGH
OVARY
62.96% LOW
32
90.63% HIGH
UPPERAERODIGESTIVETRACT
9.38% LOW
M Jurk
atDLD
1
x Jurk
atDLD
1
x AT163
6
Jurk
tDLD
1
<
AT1002isotype ctrl
(40 µg)AT1636(40 µg)
Input(~15 µg)
Coo
mas
sie
(tota
l pro
tein
s)
<<
negative control: grey filed histogram.AT1636: solid black line.
10-12 10-11 10-10 10-9 10-8 10-7
0
20
40
60
80
100
concentration [M]
spec
ific
ly
sis
[%]
DLD1
10-12 10-11 10-10 10-9 10-8 10-7
concentration [M]
HCT116
10-12 10-11 10-10 10-9 10-8 10-7
concentration [M]
A375 AT1002 TCE anti-HA Influenza; negative controlAT1636wt TCEAT1636IYN TCETCE positive control
EC₅₀:139pMMaxkill:89%
EC₅₀:476pMMaxkill:69%
EC₅₀:926pMMaxkill:91%
10-12 10-11 10-10 10-9 10-8 10-7
concentration [M]
HT29
EC₅₀:11nMMaxkill:12%
