HSA - Hilti...HSA ウ ェッジ式締付方式 金属系アンカー 技術マニュアル 更新02/2017 6 tfix HSA, HSA-R, HSA-F M6 M8 M10
800 3 900 3 900 700 100 600 15 80 500 400 MSLN.CD3.hSA 0.2 … · 2020. 11. 6. · NM21-1480...
1
NM21-1480 surrogate (NM21-1601) synergizes with MSLN.CD3.hSA to promote human lung cancer growth inhibition in vivo -20 0 20 40 60 80 100 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 [pM] Specific killing [%] 0 -20 0 20 40 60 80 100 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 [pM] Specific killing [%] 0 1 5 7 9 12 14 16 19 21 23 26 28 30 33 35 37 40 0 100 200 300 400 500 600 700 800 Days Tumor volume (mm 3 ) No treatment (no PBMCs) Palivizumab biMSLN.CD3.hSA 0.2 mg/kg biMSLN.CD3.hSA 1 mg/kg MSLN.CD3.hSA 0.2 mg/kg MSLN.CD3.hSA 1 mg/kg Concept: Selective T cell-mediated depletion of tumor cells A low affinity bivalent mesothelin-binding MATCH4 multispecific T cell engager increases cytotoxic selectivity for high mesothelin expressing cells Bithi Chatterjee, Alexandre Simonin, Daniel Snell, Tea Gunde, Christian Hess, Matthias Brock, Maria Johansson, Stefan Warmuth, Christopher Weinert, Niels Kirk, Nicole Bassler, Dania Diem, Naomi Flueckiger, Robin Heiz, Benjamin Kuettner, Dana Mahler, Diego Morenzoni, Sandro Wagen, Julia Zeberer, and David Urech Numab Therapeutics AG, Einsiedlerstrasse 34, 8820 Wädenswil, Switzerland Background: The effective treatment of solid tumors remains an unmet medical need. Several concepts exist to treat malignancies, including antibody-drug or - immunotoxin conjugates, immune checkpoint inhibition, CAR- T cells, as well as bispecific T cell engagers. CD3-based T cell engagers are highly potent therapeutic molecules with T cell cytotoxicity activities in the picomolar range. Alongside this highly potent anti-tumor activity is the risk of on-target off-tumor effects due to low levels of expression of the target antigen in normal tissue, as has been observed for the tumor- associated antigen mesothelin (MSLN). Figure 1. A. BiMSLN.CD3.hSA does not deplete healthy cells due to its low affinity binding domains and does not bind to shed soluble MSLN in circulation. B. Avidity-based binding of high MSLN expressing cells like mesothelioma, concomitant with CD3 engagement on T cells, causes the depletion of tumor cells. Conclusions and potential benefits SITC Annual Meeting 2020 - Poster # 684 www.numab.com IMPROVED THERAPEUTIC WINDOW Numab’s bivalent biMSLN.CD3.hSA MATCH4 efficiently depletes mesothelioma cells expressing high levels of MSLN and spares normal epithelial cells. The bivalent binding provides further resistance to shed soluble MSLN. SITC 2020 Poster # 684 MATCH4™: combinations for optimized potency Figure 2. A. MATCH™ (Multispecific Antibody-based Therapeutics by Cognate Heterodimerization) molecules are modular and can be combined in numerous ways and formats. The advantages include: 1. Convenient permutation of binding domains by simple plasmid exchange, 2. Multispecific format without the risk of light chain mispairing, and 3. No Fc domain requirement. B. Schematic representation of a MATCH4 molecule. C. Structural model of a MATCH4 molecule (prepared in BIOVIA Discovery Studio software). D. Representative SE-HPLC chromatogram of biMSLN.CD3.hSA. SITC 2020 Poster # 684 Non-activated T cell Healthy Cell (Pleural Epithelia) EXTRATUMORAL Mesothelioma CD3 hSA MSLN A. B. biMSLN.CD3.hSA Tumor Type No. of patients with MSLN-positive disease (%) Mesothelioma Epithelioid Sarcomatous 290 of 352 (82) 248 of 261 (95) 0 of 23 (0) Pancreatic adenocarcinoma 303 of 357 (85) Epithelial ovarian cancer High grade serous Endometroid Mucinous Clear cell 346 of 494 (70) 248 of 332 (75) 36 of 52 (69) 2 of 19 (11) 11 of 21 (52) NSCLC Adenocarcinoma Squamous 1,157 of 2,036 (57) 1,082 of 1,686 (64) 40 of 188 (21) Biliary cancer, extrahepatic 93 of 98 (95) Triple negative breast cancer 33 of 50 (66) Normal Mesothelium (MeT-5A) Lung tumor (H226) Bivalent, low affinity Monovalent, high affinity CD3 Healthy Cell Tumor cell MSLN -20 0 20 40 60 80 100 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 [pM] Specific Tumor Cell Lysis [%] 0 -20 0 20 40 60 80 100 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 [pM] Specific Tumor Cell Lysis [%] 0 ✓ Cytotoxic activity of biMSLN.CD3.hSA to MSLN-positive tumor cells is not impacted by the presence of high concentrations of soluble MSLN. Bivalent biMSLN.CD3.hSA T cell engager triggers potent tumor cell lysis and T cell activation in the presence of excess soluble MSLN (sMSLN) as compared to monovalent MSLN.CD3.hSA Larger therapeutic window for bivalent biMSLN.CD3.hSA T cell engager due to preferential avidity-based binding to tumor cells Bivalent biMSLN.CD3.hSA T cell engager actively kills tumor cells in a MSLN-dependent manner Human lung cancer growth is inhibited upon MSLN targeting Bivalent MSLN binding • Low-affinity, bivalent αMSLN domain preferentially engages MSLN-overexpressing malignant cells while sparing healthy, MSLN+ cells Extended half-life Fc-less • Avoids Fc-mediated adverse effects • Avoids internalization and degradation by macrophages • Half-life comparable to conventional IgG due to serum albumin binding domain Tumor-directed T cell stimulation • Tumor-restricted activities, unlike ADCs, which can release cytolytic agents into circulation causing dose-limiting toxicities Minimal impact of soluble MSLN • High avidity, low affinity binding to MSLN render biMSLN.CD3.hSA resistant to high concentrations of soluble MSLN present in patient blood ✓ Bivalent biMSLN.CD3.hSA shows significant inhibition of MSLN+ human HPAC pancreatic adenocarcinoma growth in humanized mice (co-injection of cancer cells and human PBMCs) ✓ Improved potency of bivalent biMSLN.CD3.hSA compared to monovalent MSLN.CD3.hSA with HPAC pancreatic xenograft expressing higher levels of MSLN Human pancreatic cancer growth is inhibited by biMSLN.CD3.hSA Human pancreatic cancer xenograft (HPAC) 1 5 8 12 15 19 22 26 29 33 36 40 44 0 100 200 300 400 500 600 700 800 900 Days Tumor volume (mm 3 ) **** all higher doses vs. palivizumab 1 5 8 12 15 19 22 26 29 33 36 40 44 0 100 200 300 400 500 600 700 800 900 Days Tumor volume (mm 3 ) ns 0.0531 ns 0.0549 * * ** ** * * Lowest doses over time Figure 3. A. Surface binding of anti-MSLN antibody to various cell types with different MSLN expression levels. B.-E. Tumor cell killing and concomitant CD8 T cell activation in the presence of biMSLN.CD3.hSA engager. H226 (B), HPAC (C), H292 (D), or normal pleural cells (E) were cocultured together with PBMCs for 40 hours and cytotoxic activity was assessed by flow cytometry. Figure 4. A. Schematic depicting preferential avidity-based binding of biMSLN.CD3.hSA. B. Comparison of cell killing on H226 tumor cells (orange line) vs. normal cells (blue line). The bivalent, low affinity biMSLN.CD3.hSA engager is depicted on the left, while the monovalent, higher affinity MSLN.CD3.hSA molecule is depicted on the right. The window of activity is shown in red (double-headed arrow). Tumor cells or normal pleural cells and PBMCs were cocultured for 40 hours and cytotoxic activity was assessed by flow cytometry. Targeting MSLN and CD3 synergizes with Numab’s next generation checkpoint modulator NM21-1480, resulting in tumor eradication Palivizumab MSLN.CD3.hSA NM21-1601 NM21-1601 + MSLN.CD3.hSA 0 5 10 15 Intratumoral CD8:CD4 ratio Day 28 * Lung cancer xenograft, H292 Lung cancer xenograft, H292 1 4 7 9 11 14 16 18 21 23 25 28 30 32 35 37 39 42 0 250 500 750 1000 1250 1500 1750 2000 Days Tumor volume (mm 3 ) ** Palivizumab MSLN.CD3.hSA 0.2 mg/kg MSLN.CD3.hSA 1 mg/kg NM21-1601 NM21-1601 + MSLN.CD3.hSA 0.2 mg/kg NM21-1601 + MSLN.CD3.hSA 1 mg/kg Table 1. Mesothelin is expressed on many types of malignancies. Frequency of patients with MSLN- positive malignancies, adapted from Hassan et al. J.Clin. Onc. 2016. MSLN expression is associated with a poor prognosis for several of these cancers, including pancreatic adenocarcinoma, biliary cancer, and breast cancer. ✓ Treatment with bivalent biMSLN.CD3.hSA significantly reduces the growth of MSLN+ human H292 lung cancer in humanized mice ✓ Similar potency to monovalent MSLN.CD3.hSA Day 40 tumor volume Human lung cancer xenograft (H292) 10 -3 10 -2 10 -1 10 0 0 150000 300000 450000 600000 750000 900000 10 0 10 1 10 2 10 3 10 4 10 5 anti-MSLN [pM] anti-MSLN MFI (Geom. Mean) H226 H292 HPAC Normal A. B. H226 Lung cancer HPAC Pancreatic cancer H292 Lung cancer A. B. Figure 5. A. Comparison of specific killing of H226 tumor cells in the presence of soluble MSLN. The bivalent, low affinity biMSLN.CD3.hSA T cell engager is depicted in orange, while the monovalent, higher affinity molecule is depicted in blue. B. Comparison of CD8 T cell activation in the presence of soluble MSLN. Tumor cells or normal cells and PBMCs were cocultured for 40 hours and the effects assessed by flow cytometry. 0.2 1 0.2 1 Ctrl No treatment 0 200 400 600 800 1000 Tumor volume (mm 3 ) MSLN.CD3 mg/kg biMSLN.CD3 mg/kg ns ns ** *** ns * * * Figure 6. A. Longitudinal tumor growth inhibition (mean + SD) in the presence of either biMSLN.CD3.hSA (orange) or MSLN.CD3.hSA (green). Immunocompromised mice were co-injected with H292 lung cancer cells and PBMCs, followed by dosing every five days. H292 expresses low levels of MSLN. B. Comparison of tumor growth inhibition on day 40. Longitudinal data were analyzed using two-way ANOVA, followed by the Tukey’s multiple comparisons test. ns: not significant; * p<0.05; ** p<0.01; *** p<0.001 A. B. Figure 7. A. Longitudinal tumor growth inhibition (mean + SD) in the presence of different doses of either biMSLN.CD3.hSA (orange) or MSLN.CD3.hSA (green). Immunocompromised mice were co-injected with HPAC pancreatic cancer cells and PBMCs, followed by dosing every five days. B. Comparison of tumor growth inhibition with the lowest doses over time. Longitudinal data were analyzed using two-way ANOVA, followed by the Tukey’s multiple comparisons test. ns: not significant; * p<0.05; ** p<0.01; *** p<0.001 Concept: Tumor-localized activation of 4-1BB combined with PD-L1 blockade Figure 8. A. NM21-1480 is a trispecific scMATCH3 molecule that binds to 4-1BB, PD-L1, and hSA. B. NM21- 1480 cannot intrinsically trigger 4-1BB clustering and signaling upon binding to 4-1BB alone. C. Clustering of 4-1BB occurs following simultaneous binding of NM21-1480 to 4-1BB+ and PD-L1+ cells, resulting in 4- 1BB signaling and concomitant blocking of the PD-1 / PD-L1 pathway. TUMOR-LOCALIZED ACTIVITY NM21-1480 leverages monovalent binding to its targets and ultra-high-affinity to PD-L1 (K D =7E-12M) in order to restrict 4-1BB signaling to the tumor microenvironment (TME), thereby allowing for safe combination of synergistic immune stimulatory activities. Healthy Cell EXTRATUMORAL NM21-1480 hSA Activated T Cell 4-1BB B. C. Figure 11. A. Longitudinal tumor growth inhibition (mean + SD) in the presence of NM21-1601 (orange), MSLN.CD3.hSA (green), or a combination of the two (red). Immunocompromised mice were co-injected with H292 lung cancer cells and PBMCs, followed by dosing every five days. H292 express low levels of MSLN and moderate levels of PD-L1. B. Comparison of CD8 infiltration between conditions. Data were analyzed using either two-way ANOVA (B) or one-way ANOVA (C), followed by the Tukey’s multiple comparisons post-hoc test. ns: not significant; * p<0.05; ** p<0.01; *** p<0.001. NM21-1601 is a surrogate for NM21-1480 and only has differences in its serum albumin binding domain to enable binding to mouse serum albumin. ✓ Remodeling of the tumor microenvironment is observed upon NM21-1601 treatment, to create a favorable environment for anti-tumor efficacy observed in combination, No tumors A. B. Activated T cell TME CD3 MSLN Tumor Cell TME 4-1BB PD-L1 NM21-1480 Inter-chain disulfide bridge Peptide linker VH2 Anti-MSLN Anti-MSLN Anti-hSA Anti-CD3 10 -10 10 -6 -20 0 20 40 60 80 100 10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 [nM] % Specific tumor cell lysis 10 -10 10 -6 -20 0 20 40 60 80 100 10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 [nM] % Specific tumor cell lysis 10 -10 10 -6 0 20 40 60 80 100 10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 [nM] CD8 + T cell activation (%) 10 -10 10 -6 0 20 40 60 80 100 10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 [nM] CD8 + T cell activation (%) Bivalent, low affinity Monovalent, high affinity A. B. Bivalent, low affinity Monovalent, high affinity Cytotoxicity Activation Normal C. -20 0 20 40 60 80 100 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 [pM] Specific killing [%] 0 -20 0 20 40 60 80 100 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 [pM] Specific killing [%] 0 0.07 pM 145 pM 2.2 pM 4-1BB is only activated in the presence of PD-L1 expression Figure 9. NM21-1480 concentration-dependent activation of 4-1BB-driven NF-κB signaling in a Jurkat reporter cell line. The assay is done in the presence of different cancer cell lines stimulated with IFNγ and expressing PD-L1 at different densities. PD-L1 density on co-cultivated cancer cells was quantified by flow cytometry. 4-1BB activation is represented as relative NF-κB activation, normalized to reference antibody Urelumab. Activated T cell TME NM21-1480 MSLN.CD3.hSA MSLN PD-L1 4-1BB CD3 Figure 10. NM21-1480 and MSLN targeting molecules such as MSLN.CD3.hSA synergize to reduce or eradicate tumor burden. Combination immunotherapy through mix and MATCH™ targeting EXTRATUMORAL 0.65 pM D. E. Fold-change Δ 0-50 ng/mL: 0.87x Δ 0-500 ng/mL: 3.9x Fold-change Δ 0-50 ng/mL: 4.6x Δ 0-500 ng/mL: 33x ✓ Potency of 4-1BB stimulation is independent of PD-L1 density. Maximal 4-1BB stimulation is correlated with PD-L1 density. PD-1 PD-1 A. B. C. D. 5 6 7 8 9 10 0 50 100 PRO2000 SE-HPLC Retention time [min] Norm. absorbance 280nm [%] biMSLN.CD3.hSA SE-HPLC Sol.MSLN Normal Mesothelium (MeT-5A) Lung tumor (H226) ✓ Bivalent biMSLN.CD3.hSA engager spares normal MSLN-expressing cells at doses where tumor cell killing is observed. A. B. Anti- 4-1BB Anti-PD-L1 Anti-hSA NM21-1480 A. Palivizumab biMSLN.CD3.hSA 0.04 mg/kg biMSLN.CD3.hSA 0.2 mg/kg biMSLN.CD3.hSA 1 mg/kg MSLN.CD3.hSA 0.04 mg/kg MSLN.CD3.hSA 0.2 mg/kg MSLN.CD3.hSA 1 mg/kg
Transcript of 800 3 900 3 900 700 100 600 15 80 500 400 MSLN.CD3.hSA 0.2 … · 2020. 11. 6. · NM21-1480...
-
NM21-1480 surrogate (NM21-1601) synergizes with MSLN.CD3.hSA
to promote human lung cancer growth inhibition in vivo
-20
0
20
40
60
80
100
10-3
10-2
10-1
100
101
102
103
104
105
[pM]
Sp
ecif
ic k
illin
g [
%]
0
-20
0
20
40
60
80
100
10-3
10-2
10-1
100
101
102
103
104
105
[pM]
Sp
ecif
ic k
illin
g [
%]
0
1 5 7 9 12 14 16 19 21 23 26 28 30 33 35 37 40
0
100
200
300
400
500
600
700
800
Days
Tu
mo
r vo
lum
e (
mm
3)
No treatment (no PBMCs)
Palivizumab
biMSLN.CD3.hSA 0.2 mg/kg
biMSLN.CD3.hSA 1 mg/kg
MSLN.CD3.hSA 0.2 mg/kg
MSLN.CD3.hSA 1 mg/kg
Concept: Selective T cell-mediated depletion of tumor cells
A low affinity bivalent mesothelin-binding MATCH4 multispecific T cell engager increases cytotoxic
selectivity for high mesothelin expressing cells
Bithi Chatterjee, Alexandre Simonin, Daniel Snell, Tea Gunde, Christian Hess, Matthias Brock, Maria Johansson, Stefan Warmuth, Christopher Weinert, Niels Kirk, Nicole
Bassler, Dania Diem, Naomi Flueckiger, Robin Heiz, Benjamin Kuettner, Dana Mahler, Diego Morenzoni, Sandro Wagen, Julia Zeberer, and David Urech
Numab Therapeutics AG, Einsiedlerstrasse 34, 8820 Wädenswil, Switzerland
AACR Annual Meeting 2019, Atlanta GA USA - Poster #1532
Background: The effective treatment of solid tumors remains an unmet medicalneed. Several concepts exist to treat malignancies, including antibody-drug or -
immunotoxin conjugates, immune checkpoint inhibition, CAR- T cells, as well as
bispecific T cell engagers. CD3-based T cell engagers are highly potent therapeutic
molecules with T cell cytotoxicity activities in the picomolar range. Alongside this highly
potent anti-tumor activity is the risk of on-target off-tumor effects due to low levels of
expression of the target antigen in normal tissue, as has been observed for the tumor-
associated antigen mesothelin (MSLN).
Figure 1. A. BiMSLN.CD3.hSA does not deplete healthy cells due to its low affinity binding domains and does not bind to shed soluble
MSLN in circulation. B. Avidity-based binding of high MSLN expressing cells like mesothelioma, concomitant with CD3 engagement on
T cells, causes the depletion of tumor cells.
Conclusions and potential benefits
SITC Annual Meeting 2020 - Poster # 684 www.numab.com
IMPROVED THERAPEUTIC WINDOW
Numab’s bivalent biMSLN.CD3.hSA
MATCH4 efficiently depletes
mesothelioma cells expressing high
levels of MSLN and spares normal
epithelial cells. The bivalent binding
provides further resistance to
shed soluble MSLN.
SITC 2020 Poster # 684
MATCH4™: combinations for optimized potency
Figure 2. A. MATCH™ (Multispecific Antibody-based Therapeutics by Cognate Heterodimerization) molecules are modular and can be combined in
numerous ways and formats. The advantages include: 1. Convenient permutation of binding domains by simple plasmid exchange, 2. Multispecific
format without the risk of light chain mispairing, and 3. No Fc domain requirement. B. Schematic representation of a MATCH4 molecule. C.
Structural model of a MATCH4 molecule (prepared in BIOVIA Discovery Studio software). D. Representative SE-HPLC chromatogram of
biMSLN.CD3.hSA.
SITC 2020 Poster # 684
Non-activated T cell
Healthy Cell
(Pleural Epithelia)
EXTRATUMORAL
Mesothelioma
CD3
hSA
MSLN
A. B.
biMSLN.CD3.hSA
Tumor TypeNo. of patients with MSLN-positive
disease (%)
Mesothelioma
Epithelioid
Sarcomatous
290 of 352 (82)
248 of 261 (95)
0 of 23 (0)
Pancreatic adenocarcinoma 303 of 357 (85)
Epithelial ovarian cancer
High grade serous
Endometroid
Mucinous
Clear cell
346 of 494 (70)
248 of 332 (75)
36 of 52 (69)
2 of 19 (11)
11 of 21 (52)
NSCLC
Adenocarcinoma
Squamous
1,157 of 2,036 (57)
1,082 of 1,686 (64)
40 of 188 (21)
Biliary cancer, extrahepatic 93 of 98 (95)
Triple negative breast cancer 33 of 50 (66)
-40
-20
0
20
40
60
80
100
10 -3 10 -2 10 -1 100 101 102 103 104 105
[pM]
Mo
no
vale
nt
MS
LN
xCD
3
Cyt
oto
xici
ty [
%]
Normal Mesothelium (MeT-5A)Lung tumor (H226)
0
Bivalent, low affinity Monovalent, high affinity
CD3
Healthy CellTumor cell
MSLN
-20
0
20
40
60
80
100
10-3
10-2
10-1
100
101
102
103
104
105
[pM]
Sp
ecif
ic T
um
or
Ce
ll L
ysis
[%
]
0
-20
0
20
40
60
80
100
10-3
10-2
10-1
100
101
102
103
104
105
[pM]
Sp
ecif
ic T
um
or
Ce
ll L
ysis
[%
]
0
✓ Cytotoxic activity of biMSLN.CD3.hSA to MSLN-positive tumor cells is not impacted by the presence of high concentrations of soluble MSLN.
Bivalent biMSLN.CD3.hSA T cell engager triggers potent tumor cell lysis and T cell activation in the presence of excess soluble MSLN (sMSLN) as compared to monovalent MSLN.CD3.hSA
Larger therapeutic window for bivalent biMSLN.CD3.hSA T cell engager due to preferential avidity-based binding to tumor cells
Bivalent biMSLN.CD3.hSA T cell engager actively kills tumor cells in a MSLN-dependent manner
Human lung cancer growth is inhibited upon MSLN targeting
Bivalent MSLN
binding
• Low-affinity, bivalent αMSLN domain preferentially engages
MSLN-overexpressing malignant cells while sparing healthy,
MSLN+ cells
Extended
half-life
Fc-less• Avoids Fc-mediated adverse effects
• Avoids internalization and degradation by macrophages
• Half-life comparable to conventional IgG due to serum
albumin binding domain
Tumor-directed T
cell stimulation
• Tumor-restricted activities, unlike ADCs, which can release
cytolytic agents into circulation causing dose-limiting
toxicities
Minimal impact
of soluble MSLN
• High avidity, low affinity binding to MSLN render
biMSLN.CD3.hSA resistant to high concentrations of soluble
MSLN present in patient blood
✓ Bivalent biMSLN.CD3.hSA shows significant inhibition of
MSLN+ human HPAC pancreatic adenocarcinoma growth in
humanized mice (co-injection of cancer cells and human
PBMCs)
✓ Improved potency of bivalent biMSLN.CD3.hSA compared to
monovalent MSLN.CD3.hSA with HPAC pancreatic xenograft
expressing higher levels of MSLN
Human pancreatic cancer growth is inhibited by biMSLN.CD3.hSAHuman pancreatic cancer xenograft (HPAC)
1 5 8 12 15 19 22 26 29 33 36 40 44
0
100
200
300
400
500
600
700
800
900
Days
Tu
mo
r vo
lum
e (
mm
3)
****
all higher doses
vs. palivizumab
1 5 8 12 15 19 22 26 29 33 36 40 44
0
100
200
300
400
500
600
700
800
900
Days
Tu
mo
r vo
lum
e (
mm
3)
ns
0.0531
ns
0.0549
*
*
**
***
*
Lowest doses over time
Figure 3. A. Surface binding of anti-MSLN antibody to various cell types with different MSLN expression levels. B.-E. Tumor cell killing and concomitant CD8 T cell activation in the presence of biMSLN.CD3.hSA
engager. H226 (B), HPAC (C), H292 (D), or normal pleural cells (E) were cocultured together with PBMCs for 40 hours and cytotoxic activity was assessed by flow cytometry.
Figure 4. A. Schematic depicting preferential avidity-based
binding of biMSLN.CD3.hSA. B. Comparison of cell killing on
H226 tumor cells (orange line) vs. normal cells (blue line).
The bivalent, low affinity biMSLN.CD3.hSA engager is
depicted on the left, while the monovalent, higher affinity
MSLN.CD3.hSA molecule is depicted on the right. The
window of activity is shown in red (double-headed arrow).
Tumor cells or normal pleural cells and PBMCs were
cocultured for 40 hours and cytotoxic activity was assessed
by flow cytometry.
Targeting MSLN and CD3 synergizes with Numab’s next generation checkpoint modulator NM21-1480, resulting in tumor eradication
Paliv
izum
ab
MSL
N.C
D3.
hSA
NM
21-1
601
NM
21-1
601
+ M
SLN.C
D3.
hSA
0
5
10
15
Intr
atu
mo
ral C
D8
:CD
4 r
ati
o
Da
y 2
8
*
Lung cancer xenograft, H292 Lung cancer xenograft, H292
1 4 7 9 11 14 16 18 21 23 25 28 30 32 35 37 39 42
0
250
500
750
1000
1250
1500
1750
2000
Days
Tu
mo
r vo
lum
e (
mm
3)
* *
Palivizumab
MSLN.CD3.hSA 0.2 mg/kg
MSLN.CD3.hSA 1 mg/kg
NM21-1601
NM21-1601 + MSLN.CD3.hSA 0.2 mg/kg
NM21-1601 + MSLN.CD3.hSA 1 mg/kg
Table 1. Mesothelin is expressed
on many types of malignancies.
Frequency of patients with MSLN-
positive malignancies, adapted
from Hassan et al. J.Clin. Onc.
2016. MSLN expression is
associated with a poor prognosis
for several of these cancers,
including pancreatic
adenocarcinoma, biliary cancer,
and breast cancer.
✓ Treatment with bivalent biMSLN.CD3.hSA
significantly reduces the growth of
MSLN+ human H292 lung cancer in
humanized mice
✓ Similar potency to monovalent
MSLN.CD3.hSA
Day 40 tumor volumeHuman lung cancer xenograft
(H292)
10-3
10-2
10-1
100
0
150000
300000
450000
600000
750000
900000
100
101
102
103
104
105
anti-MSLN [pM]
an
ti-M
SLN
MF
I (G
eo
m. M
ea
n) H226
H292
HPAC
Normal
A. B. H226 Lung cancer HPAC Pancreatic cancer H292 Lung cancer
A. B.
Figure 5. A. Comparison of specific killing of H226 tumor cells in the presence of soluble MSLN. The bivalent, low affinity biMSLN.CD3.hSA T cell engager is depicted in orange, while the monovalent, higher affinity
molecule is depicted in blue. B. Comparison of CD8 T cell activation in the presence of soluble MSLN. Tumor cells or normal cells and PBMCs were cocultured for 40 hours and the effects assessed by flow cytometry.
0.2 1
0.2 1
Ctrl
No
trea
tmen
t
0
200
400
600
800
1000
Tu
mo
r vo
lum
e (
mm
3)
MSLN.CD3
mg/kg
biMSLN.CD3
mg/kg
ns
ns
**
***
ns
*
*
*
Figure 6. A. Longitudinal tumor growth inhibition (mean +
SD) in the presence of either biMSLN.CD3.hSA (orange) or
MSLN.CD3.hSA (green). Immunocompromised mice were
co-injected with H292 lung cancer cells and PBMCs,
followed by dosing every five days. H292 expresses low
levels of MSLN. B. Comparison of tumor growth inhibition on
day 40. Longitudinal data were analyzed using two-way
ANOVA, followed by the Tukey’s multiple comparisons test.
ns: not significant; * p
