CN114634580B

CN114634580B - Development of membrane anchored IL-15 super complex and application thereof in tumor immune cell treatment

Info

Publication number: CN114634580B
Application number: CN202210275846.0A
Authority: CN
Inventors: 徐振宇; 何伟杰; 何连君
Original assignee: First Affiliated Hospital of Wannan Medical College
Current assignee: First Affiliated Hospital of Wannan Medical College
Priority date: 2022-03-21
Filing date: 2022-03-21
Publication date: 2024-01-30
Anticipated expiration: 2042-03-21
Also published as: CN114634580A

Abstract

The invention relates to the field of tumor immunotherapy, in particular to development of a membrane anchored IL-15 super complex and application thereof in the field of tumor immunotherapy, mainly comprising carrier construction of the IL-15 super complex, wherein the carrier comprises a signal peptide, a Sushi domain of IL-15-N-72D, IL-15Rα, a connecting peptide, a CD4 molecular bracket and a suicide gene R structure, and is connected with an exogenous inserted gene through a P2A connecting peptide. The invention mainly enables immune cells to continuously express IL-15, improve the quantity of TSCM, increase the function of the immune cells, enable the immune cells to keep continuous killing function, induce apoptosis when necessary, ensure the safety of treatment and have great significance in the field of clinical treatment of the immune cells through the molecular design of transmembrane and suicide gene R structures.

Description

Development of membrane anchored IL-15 super complex and application thereof in tumor immune cell treatment

Technical Field

The invention relates to the field of immune cell tumor treatment, in particular to development of a membrane-anchored IL-15 super complex and application thereof in tumor immunotherapy.

Background

Adoptive immune cell therapy is a novel anti-tumor therapeutic means in the current biomedical field after surgery, chemotherapy, radiotherapy and targeted therapy. The method mainly comprises the steps of collecting immune cells of a patient, carrying out in vitro modification, activation, culture and amplification, enhancing the targeting killing function of the cells, and then, returning the cells to the patient, thereby achieving the purpose of killing and eliminating tumor cells by enhancing the immune system function of the organism. Currently, adoptive immune cell therapies mainly include CAR-T, LAK, CIK, DC, NK and TCR, etc., among which CAR-T cell immunotherapy is the most attractive category. CAR-T (Chimeric antigen receptor T cell, chimeric antigen receptor T cells) is produced by transferring genetic material with specific antigen recognition domain and T cell activation signal into T cells by genetic modification technique, and activating T cell killing effect only when contacting TAA, and continuously killing and generating cytokine. Tumor immune cell therapy has been developed in recent years, and has achieved breakthrough progress in clinic, particularly CAR-T cells targeting CD19 antigen, and has been remarkable in treatment effect in blood tumor and lymphoma, and has achieved complete remission in some patients. This persistence is directly related to long-term immune monitoring of CAR T cells. Once the antigen is cleared in the immune response, the effects of the immune cells will disappear. Thus, there are still a number of clinical bottlenecks in current tumor immune cell therapy techniques. Numerous studies have shown that patients receiving treatment have a recurrence rate of up to 46% within 1 year after complete remission is achieved. Analysis of the patient's sub-population of immune cells and the returned CAR-T cells showed that the ratio of Tcm to Tscm in the product and the ratio of Tcm to Tscm in the subject after return was positively correlated with time of remission and maintenance of CAR-T antitumor activity. The higher the proportion of Tcm and Tscm in CAR positive T cells in CAR-T cells infused by the patient, the greater the likelihood that the patient will obtain long-term relief. Preclinical animal experiments also show that reinfusion of CAR-T cells with Tcm as the major subpopulation can significantly extend survival time of mice. The currently known Tcm preparation method is to isolate primary T cells from peripheral blood, whereas TSCM represents only a small fraction (2-3%) of Peripheral Blood Mononuclear Cells (PBMC), which is difficult to meet clinical demands even by in vitro expansion.

IL-15 is a pleiotropic cytokine with similar biological functions as IL-2, and can activate T cells, B cells and NK cells, mediate proliferation and survival of these cells, activate, maintain and expand CD8+ memory T cells, and maintain the stem properties of T cells. Does not promote induction of activated T cell apoptosis nor Treg proliferation, and cd8+ T cells are extremely sensitive to IL-15. Therefore, IL-15 is an excellent choice for anti-tumor.

IL-15Rα is a high affinity receptor for IL-15 that binds first to cell surface IL-15 and the IL-15/IL-15Rα complex must ultimately bind to two other receptor subunits, the IL-15Rβ/IL-2R subunit and the γC subunit, before it stimulates T lymphocyte proliferation. The IL-15/IL-15Rα complex and IL-15Rβ/IL-2R subunits and γC subunits are mainly expressed in two ways: one is the IL-15/IL-15Rα complex formed on the surface of activated monocytes and the like, which "presents" IL-15 from one cell to another by "trans" presentation to target cells that are adjacent to the receptors for IL-15Rβ/IL-2R and γC. Another approach is that IL-15Rα presents IL-15 "cis" to the same cell expressing IL-15Rβ/IL-2R and γC receptors, signaling within the same cell. Among them, trans-presentation is the most important. However, the trans-presentation mechanism requires that one cell presents its cell membrane-bound IL-15/IL-15Rα complex to another target cell expressing IL-15Rβ/IL-2R and γC receptors, in effect a "cell" to "cell" interaction, which requires that one cell must come into contact with another cell to effect, both as required by the body's immune system to regulate and to some extent restrict immunotherapy of related diseases (e.g., tumors, infections) using the IL-15/IL-15Rα complex.

Therefore, how to use genetic engineering technology to make cells produce membrane-anchored IL-15 super complex is a scientific and worth going into question.

Disclosure of Invention

The present invention provides membrane-anchored IL-15 super-complexes for use in immune cell therapy. The super complex can be continuously expressed on the surface of a cell membrane, contains a suicide gene R structure, has small molecular weight, greatly promotes proliferation and survival of immune cells, improves the dryness and the activity of the immune cells, and ensures the safety of products.

In order to solve the problems, the invention provides the following technical scheme:

specifically, the IL-15 super complex comprises a signal peptide, a suicide gene R structure, a Sushi domain of IL-15-M-N72D, IL-15Rα mutant of IL-15 and a CD4 molecular scaffold, and the complex is connected with an exogenous inserted gene fragment through a 2A connecting peptide.

The R structure is a CD20 epitope polypeptide, and the expression of the polypeptide on the surface of a cell can be cleared by rituximab in vivo through CDC and ADCC forms.

The IL-15 mutant IL-15-M-N72D and the "Sushi" domain of IL-15Rα in the IL-15 super complex are linked by a Linker peptide (Linker);

the IL-15 super complex anchors the CD-4 transmembrane region to the cell membrane by using the CD-4 transmembrane region as a scaffold;

the 2A connecting peptide used for connecting the IL-15 super complex and the exogenous inserted gene fragment is P2A or F2A;

specifically, the IL-15 super complex comprises: the suicide gene R structure, linker1, interleukin-15 mutant IL-15-M-N72D, linker2, the "Sushi" domain of IL-15Rα, and the CD-4 transmembrane region are connected in sequence;

the signal peptide is CD8, GM-CSF, CD4, CD28, CD137, or a mutant/modified form thereof, or a combination thereof.

Specifically, the signal peptide is a signal peptide derived from CD8, and the amino acid sequence of the signal peptide is SEQ ID NO:1 (DMWTWILFLVAAATRVHS);

specifically, the amino acid sequence of the R structure of the suicide gene is SEQ ID NO:2 (ACPNSNPSLC);

specifically, the amino acid sequence of the IL-15-M-N72D mutant of the IL-15 is SEQ ID NO:3 (NWVNVISDLKKIEDLIQSMHIDATLYTASDVHPSCKVTAMKCFLLELQVISLESGDASIHDDVENLIILANDSLSSNGNVTESGCAECEELEEKNIKEFLWSFVHIVQMFINTS);

specifically, the "Sushi" domain of IL-15 ra is the first cysteine residue (C1) following the signal peptide of IL-15 ra and terminates at the fourth cysteine residue (C4) following the signal peptide. The amino acid sequence of this "Sushi" domain is SEQ ID NO:4 (ITCPPPMSVEHADIWAKSYSLYSRERYICNSAFKRKAGTSSLTECVTNKATNVAHWTTPSLKCIRD);

specifically, the amino acid sequence of the CD-4 transmembrane domain scaffold is SEQ ID NO:5 (VNVVMRATKNTCVWGTSKMSKNKAKVSKRKAVWVNAGMWCSDSGVSNIKVTWSTVMAIVGGVAGI GGI):

specifically, the Linker peptide Linker may be (GxSy) n, wherein: g is glycine, S is serine, x is 1, 2, 3 or 4, y is 1, 2, 3 or 4, and n is 1, 2, 3, 4, 5 or 6.

Specifically, the amino acid sequence of the Linker is selected from SEQ ID NO:6 (SGGGSGGGGSGGGGSGGGGSGGGSLQ), SEQ ID NO:7 (GGGGS), SEQ ID NO:8 (GGGGSGGGGSGGGGSGGGGS), SEQ ID NO:9 (EAAAK), SEQ ID NO:10 (EAAAKEAAAKEAAAK), SEQ ID NO:11 (GSADDAKKDAAKKDGKS), SEQ ID NO:12 (SSADDAKKDAAKKDDAKKDDAKKDA);

preferably, the amino acid sequence of "Linker1" is SEQ ID NO:6, the amino acid sequence of "Linker2" is SEQ ID NO:7, preparing a base material; preferably, the 2A connecting peptide is P2A, and the amino acid sequence of the 2A connecting peptide is SEQ ID NO:13 (GSGATNFSLLKQAGDVEENPGP) a sequence shown in (d);

in another aspect, the invention provides a biological molecule vector and a host cell. The biological molecular vector and the host cell comprise nucleic acid molecules for encoding the IL-15 super complex.

In yet another aspect, the invention provides methods of using the above IL-15 super-complexes in tumor immune cell therapy.

In particular, the cells used in tumor immune cell therapy include, but are not limited to, NK cells, CAR-T cells, TCR-T, DC and the like.

Drawings

FIG. 1 IL-15 schematic diagrams of supercomplex

FIG. 2 SDS-PAGE detection result of recombinant IL-15 super complex

FIG. 3 flow cytometry detection of IL-15-CD19-CAR expression on T cell surfaces

FIG. 4 amplification curves of different types of CAR-T cells

FIG. 5 comparison of CAR-T killing efficiency of different types

FIG. 6 flow cytometry detection of TSCM-like cell expression levels

FIG. 7 IL-15 detection of the suicide function of the super-complex

Advantageous effects

Compared with the prior art, the invention has the following advantages:

(1) The complex can be continuously expressed in immune cells, is anchored on cell membranes through shorter CD-4 structural elements, does not need to add exogenous cell factor IL-15, and can lead the immune cells to continuously proliferate and survive and exert anti-tumor activity;

(2) When abnormal conditions occur, the expression of a suicide gene R structure can be started, apoptosis is induced, and safety is ensured;

(2) Each fragment is the minimum structural element necessary for functioning, and has small molecular weight;

(3) By increasing the number of TSCM, the stem property and the activity of immune cells are enhanced, so that the immune cells have longer lasting and effective functions, and the tumor recurrence of immune cell treatment can be prevented.

Detailed Description

The invention will be further illustrated in detail with reference to the following examples, which are selected from the group consisting of CD19-CAR-T cells, but are not intended to limit the invention and are merely illustrative of the invention.

EXAMPLE IL-15 super Complex Gene synthesis and vector construction

The general biological systems (Anhui) limited company construction was commissioned. The sequences were assembled in the order of the signal peptide (SEQ ID NO: 1), "suicide gene" R structure (SEQ ID NO: 2), linker1 (SEQ ID NO: 6), IL-15 mutant IL-15-M-N72D (SEQ ID NO: 3), linker2 (SEQ ID NO: 7), IL-15Rα "Sushi" domain (SEQ ID NO: 4), CD-4 transmembrane region (SEQ ID NO: 5). The genes were routinely synthesized after optimization and cloned into plasmid vectors. The IL-15 super complex pattern is shown in FIG. 1.

EXAMPLE two expression and purification of IL-15 super Complex

Transferring the expression vector into expression host bacteria (E.coli DH5 alpha), amplifying the host bacteria for about 8-10 hours, and adding 0.8mM IPTG (isopropyl-B-D-thiogalactose) to induce the expression of the plasmid. After induction for about 6 hours, the cells were collected, and after addition of a disruption buffer (pH 7.5, 20mM Tris-HCl, 50mM NaCl, 1% Triton-1 00, 20% glycerol) and ultrasonication, the supernatant was passed through a nickel column, after completion of loading, the nickel column was eluted with an eluent (pH 7.5, 20mM Tris-HCl, 50mM NaCl, 0.1% Triton X-100, 500mM imidazole, 20% glycerol) and the eluate was collected, and the collected proteins were dialyzed into 20mM Tris,50mM NaCl pH7.5, 20% glycerol, and after completion of the dialysis, ultrafiltration concentration was performed. The purified IL-15 super complex was identified by SDS-PAGE, and the results are shown in FIG. 2, wherein the molecular weight of the super complex is about 27KD, and the theoretical molecular weight of the super complex is consistent with that of the super complex in FIG. 2.

Example construction of a three CAR plasmid

The general biological systems (Anhui) limited company construction was commissioned.

The plasmid of CAR-CD19 is PST#1;

the sequences were assembled as PST#2 in the order of the "Sushi" domain of IL-15 mutant IL-15-M-N72D, IL-15Rα, fc fusion protein, P2A and CAR gene;

the plasmid in which the sequences were assembled in the order of the signal peptide, "suicide gene" R structure, linker1, IL-15 mutant IL-15-M-N72D, linker2, IL-15Rα "Sushi" domain, CD-4 transmembrane region, P2A and CAR gene was PST #3.

EXAMPLE four packaging of lentiviral vectors

(1) Preparation of 293T cells: 24 hours before transfection, 10cm dishes, 9X 10 were spread ⁶ Cells/dish (10 ml); when the cell fusion degree reaches 80-90%, the cell can be used for transfection;

(2) and (3) virus packaging: the expression plasmid and helper plasmid were added in total at 22.5ug to transfection at the ratio indicated in Table one.

TABLE I addition ratio of plasmids

(3) After 293T cell exchange, the plasmid solution prepared above was mixed with Lipo8000 and transferred to 293T cell culture medium, gently mixed, and cultured in 5% CO2 cell incubator at 37 ℃. After 8h of infection, a new serum-added DMEM medium was changed; after 48h post infection the supernatant was collected and stored at 4 ℃. And the same volume of serum-containing DMEM medium was added, the supernatant was collected again 72 hours after infection, the virus supernatant collected twice was centrifuged at 3000rpm at 4℃for 10 minutes, cell debris was removed, and the supernatant was filtered through a 0.45 μm filter membrane. The pre-prepared virus extract is added into a 50mL centrifuge tube, then slow virus supernatant is slowly dripped onto the virus extract to form layering (the volume ratio of the virus extract to the virus supernatant is 1:4), the virus extract is centrifuged for 4 hours at the temperature of 4 ℃ and the precipitate is resuspended after centrifugation, and the obtained precipitate is placed into a 1.5mL centrifuge tube. The virus titer was detected by QPCR and the virus was stored at-80℃for a long period of time.

As a result of analysis, the virus titer of the packaged virus is obviously higher than that of the virus packaged by the CAR-CD19 plasmid according to the design scheme in the patent. The viral expression of CD4 as an IL15 super complex of the transmembrane region was also significantly increased compared to the Fc transmembrane region.

Table two, results of virus titer detection

Group of	Virus titre TU/ml
		A	2.1*10 ⁸
B	5.9*10 ⁹
		C	9.2*10 ⁹

Example five isolation of immune cells to be engineered in human peripheral blood

(1) Human peripheral blood was obtained from healthy donors, after which 20ml of lymphocyte separation fluid was first added to a 50ml centrifuge tube, and then 20ml of whole blood was carefully added along the tube wall, ensuring that stratification was evident. Slowly rising and slowly falling, centrifuging at 650g for 20min, sucking a white membrane layer into a 50ml centrifuge tube, adding PBS, washing, discarding supernatant, re-suspending by using RPMI 1640 medium, separating CD3 positive T cells by using the beads of the CD3 antibody, and culturing group A by adding cell stimulating factors CD3, CD28 and IL-2 with the final concentrations of 200ng/ml, 200ng/ml and 40ng/ml respectively. The final concentrations of the combination B and the group C added with CD3, CD28, IL-7 and IL-21 are respectively 200ng/mL, 10ng/mL and 20ng/mL.

(2) After 24 hours, the pre-packaged A, B, C three groups of viruses were tested at an MOI of 100, i.e., lentiviruses: t cells = 100:1, cultured in a 5% co2 incubator at 37 ℃, different types of CAR-T cells infected with virus were obtained.

Example six flow cytometry detection of expression of IL-15 on T cell surface after infection

The expression of IL-15 on the surface of CAR-T cells infected with A, B, C viruses was detected by flow cytometry using a Green Fluorescent Protein (GFP) carried by the lentiviral vector itself as a screening marker. CAT-T cells were centrifuged at 2000rpm for 5min and resuspended in PBS. 100ul of cell resuspension is taken and checked on a machine. The results are shown in FIG. 3, and the expression level of IL-15 in group C is higher than that in group A, B.

Example seven in vitro proliferation and maintenance Capacity of IL-15 secreting CD19CAR-T cells

After T cell infection with the A, B, C three lentiviruses, CAR-T cell number statistics were performed by microscopic counting at time points of days 2, 4, 6, 8, 10, 12, 14, 16, respectively. The number of CAR positive cells was calculated using flow cytometry, with CD3 molecules as gating, using antibody-coupled fluorescent molecules that specifically recognize anti-CD19scFv for flow labeling. A. The CAR-T cell expansion proliferation curves prepared by infection with three groups of B, C lentiviruses are shown in fig. 4. As can be seen from the figure, the level of expansion of CAR-T cells in group C was significantly increased.

Example eight cell killing experiment effect evaluation

(1) Culturing K562 cells capable of expressing CD19 and T cells separated from peripheral blood respectively;

(2) 3 days before the start of the experiment, the virus of group A, B and C was used to infect T cells isolated from peripheral blood according to MOI of 100, and the infected cells were placed at 37℃with 5% CO ₂ After 96 hours of medium culture, performing a cell killing experiment;

(3) Collection of target cells (CD19+K562) 1.0X10 ⁶ cells and effector cells (T cells infected in step 2) were 1.5X10 each ⁶ After centrifugation at cells,2000rpm for 6min, the supernatant was discarded and the pellet was resuspended in 1mL of 1 XPBS solution, respectively.

(4) After centrifugation at 2000rpm for 6min again, the supernatant was discarded.

(5) Effector cells were resuspended in 700 μl of medium (1640 medium+10% FBS) and target cells were each resuspended in 1mL of medium (1640 medium+10% FBS);

(6) Setting experimental holes with the effective target ratio of 1:1 and 10:1, and setting a control group, wherein each group comprises 3 compound holes;

(7) Centrifuging the plate under the conditions of 250 Xg and 5min, placing the plate in an incubator with 5% CO2 at 37 ℃ for 24 hours, and centrifuging the plate again with 250 Xg and 5 min;

(8) 50. Mu.L of supernatant was taken into a new 96-well plate and 50. Mu.L of substrate solution (should be handled in the dark) was added to each well;

(9) Incubating for 25-30min in dark;

(10) Add 50. Mu.L of stop solution per well;

(11) Detecting absorbance at 490nm by an enzyme-labeled instrument;

(12) Taking the average value of 3 compound holes; subtracting the average value of the absorbance values of the background of the culture medium from the absorbance values of all experimental holes, target cell holes and effector cell holes; subtracting the mean value of the volume correction control absorbance value from the absorbance value of the maximum value of the target cells;

(13) The corrected values obtained in step 12 are taken into the following formula, and the percent cytotoxicity produced for each target ratio is calculated, where killing efficiency = (experimental well-effector cell well-target cell well)/(target cell maximum well-target cell well) ×100%. As shown in fig. 5, T cells were control group, and the killing effect of CAR-T cells of group C on target cells was better compared with those of A, B, C group, indicating that cells containing IL15-CAR-CD19 super complex could enhance the killing ability of effector cells.

EXAMPLE nine C group CAR-T cell expression level of TSCM-like cells

After T cells were individually infected with A, B, C lentiviruses, TSCM-like cell number analysis was performed on day 12 using flow cytometry, and CD45RA+CD45RO-CCR7+CD95+ cells were selected and the results are shown in FIG. 6. From the results, it can be seen that the T cells after group C lentivirus infection had a higher number of TSCM-like cells. CD 19-specific CAR-T cells produced by membrane-bound IL-15 have long-term persistence and excellent anti-tumor activity in vivo.

Example ten IL-15 super Complex suicide function detection

1. The A, B, C group of CAR-T cells were validated to achieve "suicide" via the CDC pathway. A. After incubation of B, C groups of CAR-T cells with 25% young rabbit complement and rituximab at different concentrations (100 ug/ml and 200 ug/ml) for 4 hours, samples were stained with annexin v/PI and apoptosis of B, C groups of CAR-T cells was assessed by flow cytometry analysis.

2. The B, C group of CAR-T cells were validated for "suicide" via the ADCC pathway. NK cells from the same donor were taken as effector cells, and after incubating effector cells, target cells and 100ug/ml rituximab for 48 hours at an effective target ratio of 8:1 and 16:1, samples were stained with Annexin V/PI and apoptosis in B, C groups of CAR-T cells were assessed by flow cytometry analysis.

The results of figure 7 show that the CAR-T cells of group C can achieve "suicide" by the CDC pathway and ADCC pathway, whereas the A, B two groups have no suicide function.

Sequence listing

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Claims

1. A membrane anchored IL-15 super complex characterized by: the IL-15 super complex is a signal peptide, a suicide gene R structure, a connecting peptide 1, a Sushi domain of IL-15-N72D, IL-15 Ralpha of IL-15 mutant, a connecting peptide 2 and a CD4 transmembrane region molecular scaffold which are sequentially connected, and the IL-15 complex is anchored on a cell membrane through the CD4 scaffold; wherein the signal peptide is a signal peptide derived from CD8, and the amino acid sequence is shown in SEQ ID NO:1 is shown in the specification; the suicide gene R structure is a CD20 epitope polypeptide, the expression of the suicide gene R structure on the surface of a cell can be cleared by rituximab in vivo through CDC and ADCC forms, and the amino acid sequence of the R structure is shown as SEQ ID NO:2 is shown in the figure; the amino acid sequence of the IL-15 mutant IL-15-N72D is shown in SEQ ID NO:3 is shown in the figure; the amino acid sequence of the "Sushi" domain of IL-15Rα is shown in SEQ ID NO:4 is shown in the figure; the amino acid sequence of the CD4 transmembrane domain scaffold is shown in SEQ ID NO:5 is shown in the figure; the amino acid sequence of the connecting peptide 1 is shown in SEQ ID NO:6 is shown in the figure; the amino acid sequence of the connecting peptide 2 is shown in SEQ ID NO: shown at 7.

2. A lentiviral expression vector, characterized in that: a nucleic acid molecule comprising a nucleic acid encoding the membrane-anchored IL-15 super complex of claim 1.

3. An immune cell capable of secreting an IL-15 complex, characterized by: infecting the immune cells with a virus comprising a nucleic acid molecule encoding the IL-15 complex of claim 1 and an exogenous insertion gene sequence; the immune cells are selected from CAR-T, NK, TCR-T, DC cells.

4. A pharmaceutical composition for immunotherapy comprising the membrane-anchored IL-15 super complex of claim 1, or the vector of claim 2, or the immune cell of claim 3.

5. Use of a membrane-anchored IL-15 super complex of claim 1, or a vector of claim 2, or an immune cell of claim 3, in the manufacture of a medicament for inducing immune cell proliferation and/or tumor treatment; the tumor is blood or solid tumor; the cells are selected from the group consisting of CAR-T, NK, TCR-T, DC cells.

6. The use according to claim 5, wherein the nucleic acid molecule encoding the membrane-anchored IL-15 super complex is linked to the foreign insert gene sequence by P2A; the amino acid sequence of P2A is shown in SEQ ID NO: shown at 13.

7. The construction method of the membrane anchored IL-15 super complex expression vector is characterized by comprising the following steps:

(1) Constructing a nucleic acid molecule encoding the membrane-anchored IL-15 super complex of claim 1;

(2) Connecting the nucleic acid molecule obtained in the step (1) with CAR-CD19 through P2A to construct a lentiviral vector, wherein the amino acid sequence of P2A is SEQ ID NO: 13;

(3) And (3) carrying out virus packaging on the obtained lentiviral vector by using 293T cells, respectively after 48h and 96h, collecting virus supernatant, and concentrating to obtain the virus vector for expressing the IL-15 complex and the CAR-CD 19.